Process of obtaining thylakoids from photosynthetic organisms; plant fractions obtained from the process; pure thylakoids; and methods of use of thylakoids as ROS scavengers, photo-protectors, biosensors, biofilters and bioreactors

ABSTRACT

This invention relates to a process by which an extract comprising integral thylakoids is obtained. The resulting extract is a potent dynamic antioxidant useful as a ROS (reactive oxygen species) scavenger. This extract is intended to be used for the treatment or prevention of diseases involving the generation of ROS, such as inflammatory diseases or cancer. This extract also finds a use as a solar screen because of its capacity to capture UV radiations and to dissipate the solar energy into heat.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a national phase filing of Patent CooperationTreaty Application PCT/CA00/01541, which was filed on Dec. 29, 2000,which in turn claims priority from Canadian Application 2,293,852 filedon Dec. 30, 1999, which is incorporated herein by reference in itsentirety for all purposes.

Process of obtaining thylakoids from photosynthetic organism; plantfractions obtained from the process; pure thylakoids; and methods of useof thylakoids as ROS scavengers, photo-protectors, biofilters andbioreactors.

FIELD OF THE INVENTION

This invention relates to the isolation and recovery of thylakoids,which are present substantially in their integral and natural state, atleast a portion of which is functional or activable. This invention alsorelates to the obtention of other soluble and insoluble plant fractionsobtained upon the isolation of thylakoids. This invention furtherrelates to the use of thylakoids as ROS scavengers, as photoprotectors,particularly against U.V. radiations, as well as biosensors, biofiltersor bioreactors.

BACKGROUND OF THE INVENTION

Antioxidants have become increasingly popular, namely in the biomedicalfield, because of their capacity to prevent the formation and thenoxious activity of reactive oxygen species (ROS).

Plants and other photosynthetic organisms are particularly well adaptedto resist the effect of ROS, especially to protect vital organellephotosynthetic membranes called thylakoids against oxidative damages andthe noxious action of U.V. radiations.

Sunlight plays a much larger role in our sustenance than we may expect:all the food we eat and all the fossil fuel we use is a product ofphotosynthesis, which is the process that converts energy in sunlight tochemical forms of energy that can be used by biological systems.Photosynthesis is carried out by many different organisms, ranging fromplants to bacteria. The best known form of photosynthesis is the onecarried out by higher plants and algae, as well as by cyanobacteria andtheir relatives, which are responsible for a major part ofphotosynthesis in oceans. All these organisms convert CO₂ (carbondioxide) to organic material by reducing this gas to carbohydrates in arather complex set of reactions. Electrons for this reduction reactionultimately come from water, which is then converted to oxygen andprotons. Energy for this process is provided by light, which is absorbedby pigments (primarily chlorophylls and carotenoids). Chlorophyllsabsorb blue and red light and carotenoids absorb blue-green light, butgreen and yellow light are not effectively absorbed by photosyntheticpigments in plants; therefore, light of these colors is either reflectedby leaves or passes through the leaves.

Other photosynthetic organisms, such as cyanobacteria (formerly known asblue-green algae) and red algae, have additional pigments calledphycobilins that are red or blue and that absorb the colors of visiblelight that are not effectively absorbed by chlorophyll and carotenoids.Yet other organisms, such as the purple and green bacteria (which, bythe way, look fairly brown under many growth conditions), containbacteriochlorophyll that absorbs in the infrared, in addition to in theblue part of the spectrum. These bacteria do not evolve oxygen, butperform photosynthesis under anaerobic (oxygen-less) conditions. Thesebacteria efficiently use infrared light for photosynthesis. Infrared islight with wavelengths above 700 nm that cannot be seen by the humaneye; some bacterial species can use infrared light with wavelengths ofup to 1000 nm. However, most pigments are not very effective inabsorbing ultraviolet light (<400 nm), which also cannot be seen by thehuman eye. Light with wavelengths below 330 nm becomes increasinglydamaging to cells, but virtually all light at these short wavelengths isfiltered out by the atmosphere (most prominently the ozone layer) beforereaching the earth. Even though most plants are capable of producingcompounds that absorb ultraviolet light, an increased exposure to lightaround 300 nm has detrimental effects on plant productivity.

Photosynthetic pigments come in a huge variety: there are many differenttypes of (bacterio)chlorophyll, carotenoids, and phycobilins, differingfrom each other in their precise chemical structure. Pigments generallyare bound to proteins, which provide the pigment molecules with theappropriate orientation and positioning with respect to each other.Light energy is absorbed by individual pigments, but is not usedimmediately by these pigments for energy conversion. Instead, the lightenergy is transferred to chlorophylls that are in a special proteinenvironment where the actual energy conversion event occurs: the lightenergy is used to transfer an electron to a neighboring pigment.Pigments and protein involved with this actual primary electron transferevent together are called the reaction center. A large number of pigmentmolecules (100–5000), collectively referred to as antenna, “harvest”light, capture photons, and transfer the light energy to the samereaction center. The purpose is to maintain a high rate of electrontransfer in the reaction center, even at lower light intensities. Thedenomination P680 is assigned to the chlorophyll pigments of thereaction center PSII, because the pair of chlorophylls entering itcomposition absorbs light mostly at a 680 nm wavelength.

Many antenna pigments transfer their light energy to a single reactioncenter by having this energy transfer to another antenna pigment, andyet to another, etc., until the energy is “trapped” in the reactioncenter. Each step of this energy transfer must be very efficient toavoid a large loss in the overall transfer process, and the associationof the various pigments with proteins ensures that transfer efficienciesare high by having appropriate pigments close to each other, and byhaving an appropriate molecular geometry of the pigments with respect toeach other. An exception to the rule of protein-bound pigments are greenbacteria with very large antenna systems: a large part of these antennasystems consists of a “bag” (named chlorosome) of up to several thousandbacteriochlorophyll molecules that interact with each other and that arenot in direct contact with protein. Chlorophyll is used by allphotosynthetic organisms as the link between excitation energy transferand electron transfer. Of particular note is the rate with which thesetransfer reactions need to occur. As the lifetime of the excited stateis only several nanoseconds (1 nanosecond (ns) is 10⁻⁹ s), afterabsorption of a quantum, energy transfer and charge separation in thereaction center must have occurred within this time period. Energytransfer rates between pigments are very rapid, and charge separation inreaction centers occurs in 3–30 picoseconds (1 picosecond (ps) is 10⁻¹²s). Subsequent electron transfer steps are significantly slower (200ps–20 ms) but, nonetheless, the electron transport chain is sufficientlyfast that at least a significant part of the absorbed sunlight can beused for photosynthesis. The pigments have a specific organisation whichshould be preserved upon isolation and purification of thylakoids if themaintenance of the function of the latter is sought.

In many systems the size of the photosynthetic antenna is flexible, andphotosynthetic organisms growing at low light (in the shade, forexample) generally will have a larger number of antenna pigments perreaction center than those growing at higher light intensity. However,at high light intensities (for example, in full sunlight) the amount oflight that is absorbed by plants exceeds the capacity of electrontransfer initiated by reaction centers. Plants have developed means toconvert some of the absorbed light energy to heat rather than to use theabsorbed light necessarily for photosynthesis. However, in particularthe first part of photosynthetic electron transfer in plants is rathersensitive to overly high rates of electron transfer, and part of thephotosynthetic electron transport chain may be shut down when the lightintensity is too high; this phenomenon is known as photoinhibition.

The initial electron transfer (charge separation) reaction in thephotosynthetic reaction center sets into motion a long series of redox(reduction-oxidation) reactions, passing the electron along a chain ofcofactors and filling up the “electron hole” on the chlorophyll, muchlike in a bucket brigade. All photosynthetic organisms that produceoxygen have two types of reaction centers, named photosystem II andphotosystem I (PS II and PS I, for short), both of which arepigment/protein complexes that are located in specialized membranescalled thylakoids. In eukaryotes (plants and algae), these thylakoidsare located in chloroplasts (organelles in plant cells) and often arefound in membrane stacks (grana). Prokaryotes (bacteria) do not havechloroplasts or other organelles, and photosynthetic pigment-proteincomplexes either are in the membrane around the cytoplasm or ininvaginations thereof (as is found, for example, in purple bacteria), orare in thylakoid membranes that form much more complex structures withinthe cell (as is the case for most cyanobacteria).

All the chlorophyll in oxygenic organisms is located in thylakoids, andis associated with PS II, PS I, or with antenna proteins feeding energyinto these photosystems. PS II is the complex where water splitting andoxygen evolution occurs. Upon oxidation of the reaction centerchlorophyll in PS II, an electron is pulled from a nearby amino acid(tyrosine) which is part of the surrounding protein, which in turn getsan electron from the water-splitting complex. From the PS II reactioncenter, electrons flow to free electron carrying molecules(plastoquinone) in the thylakoid membrane, and from there to anothermembrane-protein complex, the cytochrome b₆f complex. The otherphotosystem, PS I, also catalyzes light-induced charge separation in afashion basically similar to PS II: light is harvested by an antenna,and light energy is transferred to a reaction center chlorophyll, wherelight-induced charge separation is initiated. However, in PS I electronsare transferred eventually to NADP (nicotinamide adenosine dinucleotidephosphate), the reduced form of which can be used for carbon fixation.The oxidized reaction center chlorophyll eventually receives anotherelectron from the cytochrome b₆f complex. Therefore, electron transferthrough PS II and PS I results in water oxidation (producing oxygen) andNADP reduction, with the energy for this process provided by light (2quanta for each electron transported through the whole chain).

Electron flow from water to NADP requires light and is coupled togeneration of a proton gradient across the thylakoid membrane. Thisproton gradient is used for synthesis of ATP (adenosine triphosphate), ahigh-energy molecule. ATP and reduced NADP that resulted from the lightreactions are used for CO₂ fixation in a process that is independent oflight. CO₂ fixation involves a number of reactions that is referred toas the Calvin-Benson cycle. The initial CO₂ fixation reaction involvesthe enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO),which can react with either oxygen (leading to a process namedphotorespiration and not resulting in carbon fixation) or with CO₂. Theprobability with which RuBisCO reacts with oxygen vs. with CO₂ dependson the relative concentrations of the two compounds at the site of thereaction. In all organisms CO₂ is by far the preferred substrate, but asthe CO₂ concentration is very much lower than the oxygen concentration,photorespiration does occur at significant levels. To boost the localCO₂ concentration and to minimize the oxygen tension, some plants(referred to as C₄ plants) have set aside some cells within a leaf(named bundle-sheath cells) to be involved primarily in CO₂ fixation,and others (named mesophyll cells) to specialize in the light reactions:ATP, CO₂ and reduced NADP in mesophyll cells is used for synthesis of4-carbon organic acids (such as malate), which are transported to bundlesheath cells. Here the organic acids are converted releasing CO₂ andreduced NADP, which are used for carbon fixation. The resulting 3-carbonacid is returned to the mesophyll cells. The bundle sheath cellsgenerally do not have PS II activity, in order to minimize the localoxygen concentration. However, they retain PS I, presumably to aid inATP synthesis.

Thylakoid organization is very sophisticated in order to extract theenergy from light, and to transfer this energy to a proper location,and/or dissipate the same. The transfer is rendered possible andefficient by separating electrical charges and a high capacity toregenerate a neutral electrical state, ready for undertaking again achange in charges (Blankenship et al. 1998).

The electron transfer between the above five main components isextremely rapid: the transfer from an activated P680 to pheophytin takesless than one picosecond. The electron transfer stops when all thepigments return to a neutral electrical charge, ready to undertake a newcycle.

Electrons are finally directed to a coupling factor to reduce NADPH,necessary in ATP synthesis, which will serve in sugar synthesis.

The term “thylakoids” is used hereinbelow and means to cover organizedphotosynthetic membrane components obtained from photosyntheticorganisms, eucaryotic and prokarytotic. When the organism haschloroplasts, the thylakoids comprise the following membraneconstituents: PSII, cytochromes b₆ and f, PSI and the coupling factor.Where thylakoids integrity and functionality has been tested from plantmaterial, it has been measured between two reference points: proximal toPSII and distal to the coupling factor. For certain applications,thylakoids do not need to be active although they are apparentlyintegral. Such thylakoids are performing and at least as stable as anyother antioxidant. Therefore, “active thylakoids” means thylakoidshaving the capacity to activate upon hydration, as opposed to inactivethylakoids which are integral but which have been actively or passivelyinactivated. In this case, the reaction center is inactive althoughthylakoids structure is substantially preserved. The “inactive”thylakoids are therefore suitable antioxidants although they do not havethe same dynamism nor do they have the same capability to regenerate, orthe same capacity to respond to ROS as the active/activable form.

Photosynthesis comprises two fundamental processes that can besummarized in the two following reactions:

CO₂+ATP+NADPH+H⁺→(CH₂O)_(n)+ADP+Pi+NADP⁺  (2)

During the first reaction in the presence of light, protons are takenfrom chloroplast water to produce ATP. The second reaction consists inusing NADPH and ATP in a series of reactions that lead to the reductionof carbon anhydride in glucides, mainly starch. These two reactionsoccur simultaneously; products formed by process (1) are directed intothe reaction of process (2). Globally, the photosynthesis results intothe production of sugars in the form of starch and sucrose and energyunder the form of ATP molecules:

Light activation follows a certain pathway in the thylakoids. Light isfirst collected by light antenna (LHCII), and the energy is directed toreaction center (PSII) and, finally to PSI which also has an independentlight collector (LHCI). Thylakoids have for functions to collect lightand to transfer light energy to a proper location for furtherphotosynthesis. The synthesis of ATP and of sugars does not take placein the thylakoids but in other chloroplast compartments.

Chlorophylls are the main active pigments. The carotenoids have morethan one role, depending on their location. A first role is as lightcollectors, which results in energy transfer from carotenoids tochlorophylls. A second role is as photoprotectors, this time the energytransfer occurring in an opposite direction between chlorophylls andcarotenoids. Carotenoid singlet state has more energy that a singletchlorophyll while, on the opposite, carotenoid triplet state has lessenergy than triplet chlorophylls. The energy states having a naturaltendency to go from a high to a low energy level, one will appreciatethat the singlet carotenoid mostly acts as a light collector passinglight energy to a singlet chlorophyll molecule while the tripletchlorophyll will readily transfer its energy to the triplet carotenoid,when the latter acts as a photoprotector in the reaction center.Carotenoids take different configurations upon associating with antennaor reaction center, which configuration may be responsible for theirenergy state upon activation. A “cis” configuration is associated withphotoprotection in the reaction center. An “all-trans” configuration isassociated with the light collector function of the antenna.

The transfer of energy is efficient only in conditions in which thepigments are very close to each other and in a specific organisation. Itis therefore very important not to disturb the natural organisation ofthe pigments, keeping the membranes in an integral state, if one wantsto purify active or fully activable thylakoids.

One advantage of recovering intact thylakoids is found in their capacityto handle ROS. Such ROS are intended to cover free radicals (includingsuper oxides), as well as non-radical oxidants such as singlet oxygen(¹O₂) and peroxides. A good review of the definition and origin of thesespecies is found in the international publication WO 94/13300. Thecontents of all the references cited hereinabove and below areincorporated herein by reference.

Free radicals are atoms, ions, or molecules that contain an unpairedelectron. Free radicals are usually unstable and exhibit shorthalf-lives. Elemental oxygen is highly electronegative and readilyaccepts single electron transfers from cytochromes and other reducedcellular components; a portion of the O₂ consumed by cells engaged inaerobic respiration is univalently reduced to superoxide radical (.O₂⁻). Sequential univalent reduction of .O₂ ⁻ produces hydrogen peroxide(H₂O₂), hydroxyl radical (.OH), and water.

Free radicals can originate from many sources, including aerobicrespiration, cytochrome P-450-catalyzed monooxygenation reactions ofdrugs and xenobiotics (e.g., trichloromethyl radicals, CCl₃, formed fromoxidation of carbon tetrachloride), and ionizing radiation. For example,when tissues are exposed to gamma radiation, most of the energydeposited in the cells is absorbed by water and results in scission ofthe oxygen-hydrogen covalent bonds in water, leaving a single electronon hydrogen and one on oxygen creating two radicals H and OH. Thehydroxyl radical, .OH, is the most reactive radical known in chemistry.It reacts with biomolecules and sets off chain reactions and caninteract with the purine or pyrimidine bases of nucleic acids. Indeed,radiation-induced carcinogenesis may be initiated by free radicaldamage. Also for example, the “oxidative burst” of activated neutrophilsproduces abundant superoxide radical, which is believed to be anessential factor in producing the cytotoxic effect of activatedneutrophils. Reperfusion of ischemic tissues also produces largeconcentrations of oxyradicals, typically superoxide. Moreover,superoxide may be produced physiologically by endothelial cells forreaction with nitric oxide, a physiological regulator, formingperoxynitrite, ONOO⁻ which may decay and give rise to hydroxyl radical,.OH. Additional sources of oxyradicals are “leakage” of electrons fromdisrupted mitochondrial or endoplasmic reticular electron transportchains, prostaglandin synthesis, oxidation of catecholamines, andplatelet activation. Many free radical reactions are highly damaging tocellular components; they crosslink proteins, mutagenize DNA, andperoxidize lipids. Once formed, free radicals can interact to produceother free radicals and non-radical oxidants such as singlet oxygen(¹O₂) and peroxides.

Singlet oxygen is a particularly noxious compound involved in theinitiation or in the perpetuation of many diseases or disorders. Thesinglet oxygen is also involved in the degradation of protein likechlorophylls. This is why a photoprotection conferred by the presence ofcarotenoids becomes important. Carotenoids protect the chlorophyll lifeand activity, they further protect the integrity of the membranes bypreventing protein denaturation. Carotenoids are capable of capting theenergy of triplet chlorophyll molecules; they become triplet carotenoidmolecules, which regenerate themselves while dissipating heat therebyavoiding the accumulation of a triplet chlorophyll, and minimizing thechances to degrade the chlorophyll.

However, in the presence of excess light, damage may occur, which mayoriginate from the formation of chlorophyll in “triplet state”. In atriplet state two electrons in the outer shell have identical ratherthan opposite spin orientation. This triplet chlorophyll readily reactswith oxygen, leading to the very reactive singlet oxygen, which candamage proteins. To counter this damaging reaction, carotenoids areusually present in close vicinity to chlorophylls. Many carotenoidsefficiently “quench” triplet states of chlorophyll, thus avoidingformation of singlet oxygen. Chlorophyll in its free form is very toxicin the light in the presence of oxygen, because a close interaction withcarotenoids is not always available under such circumstances. Therefore,all chlorophyll in a cell in aerobic organisms is bound to proteins,generally with carotenoids bound to the same protein.

A major difficulty in measuring enzyme kinetics at relatively short timescales (less than 1 ms) is that “traditional” enzyme reactions require amixing of substrate and enzyme, which usually takes a relatively longtime. Kinetic analysis of light-driven reactions such as photosyntheticelectron transport have a great advantage in this respect: reactions canbe triggered simply by a light pulse, which can be even shorter than 1ps. Moreover, many of the components participating in electron transferhave different absorption spectra depending on whether they are in theoxidized or reduced form. Using laser spectroscopy methods or morestandard optical spectroscopy, it is relatively simple to follow theelectron around on a timescale between 1 ps and several ms. The primarycharge separation occurs in several ps, and reactions become graduallyslower as they involve components that are further away from thereaction center. Because of the fast speed of early reactions, theelectron and the “electron hole” are physically separated rapidly by alarge distance (the electron generally has traveled about 2 nm to theother side of the membrane within 1 ns after charge separation), so thatback reactions (charge recombinations) are not favorable anymore.Unpaired electrons on reactants that are transiently formed during redoxreactions involving transfer of a single electron in many instances canbe detected using electron paramagnetic resonance (EPR) and derivedtechniques (including ENDOR, electron nuclear double resonance, andESEEM, electron spin echo envelope modulation). Many of these techniquescan be used to kinetically follow redox reactions, and provide detailedinformation regarding electron spin distributions etc. Therefore,photosynthetic membranes and reaction centers have a prominent place asexperimental systems in biochemistry and biophysics.

The anti-oxidative potential of a compound such as chlorophyll isexemplified in equation (1)³Chl*+³O₂→Chl+¹O₂*  (1)

Chlorophyll that has been excited into presence of oxygen becomes in atriplet state (³Chl*), and disactivates to return to a fundamental stateby producing singlet oxygen (a noxious species) in cells. Plants havefound an efficient means by which they can solve the problem ofoverproduction of singlet oxygen. The plants transfer the chlorophyllenergy to another pigment which has an inferior energy state. Thatpigment called carotenoid (equation 2) is abundant in plants.³Chl*+Car→Chl+³Car*  (2)

Although triplet chlorophyll has more energy than a correspondingcarotenoid, the converse is true for the singlet state. As shown inequations 3a and 3b, an activated singlet carotenoid transfers itsenergy to a chlorophyll molecule which becomes activated in a singletstate.Car+energy→¹Car*  (3a)¹Car*+Chl→Car+¹Chl*  (3b)

Carotenoids in a triplet state desactivates without forming a noxiousoxygen species. Equation 4 shows that carotenoids inactivate byreturning to a fundamental state and by heat dissipation.³Car*→Car+Heat  (4)

It appears that it is important not to produce ROS to preserve theproperties of the pigments in an extract, but it is also important toremove those ROS that may be generated during isolation. For achievingthis, we have given favor to a way to reverse the equilibrium ofequation 1. Consequently, the converse equation 1 is found in equation5.Chl+¹O₂*→³Chl*+³O₂  (5)

To avoid reversal of equation 5, activated triplet chlorophyll moleculeneeds to be in close contact with a carotenoid in its fundamental state,which takes the transferred energy and dissipates the latter as heat.This way the reversibility of equation 5 is restricted insofar aschlorophyll and carotenoid pigments can be found in very close proximityso as to transfer to one another their energy.

From the above equation 5, it is apparent that, to obtain an extractthat is optimally active, it is preferable to take every possiblemeasures to maintain both pigments (chlorophyll and carotenoid) in theirfundamental state. Isolated carotenoids, e.g. carotenoids not organizedin thylakoid structures, would not be capable of an efficient quenchingof triplet chlorophyll molecules. The advantage of having organizedpigments is that the extract will retain the dynamism of naturalthylakoid membranes, which confers to them the capacity to capture ROS,to transfer the energy and to return to a state capable of undertakingnew activation cycles again. This dynamism and capacity to regenerate isunique to organized pigments. It is important to mention that the abovereactions are spontaneously produced and this, in absence of light. Thisobservation is important from a therapeutic point of view, becauseinternal administration of a thylakoid extract would preclude thepresence of light.

Thylakoids having optimized configuration and carotenoid proportionswill retain full activity especially toward ROS. Such an antioxidantwill be useful to reduce the expression of diseases or disorders thatinvolve the production of ROS. Such diseases or disorders can be thosewith an etiology related to inflammation, cancer and contact withradiations. Such diseases or disorders comprise those affecting Skin:such as bums, solar radiation, psoriasis, dermatitis; Brain: such astrauma, stroke, Parkinson, neurotoxins, dementia, Alzheimer; Joints:such as rheumatoid arthritis and arthrosis; Gastrointestinal tract: suchas diabetes, pancreatitis, endotoxin liver injury, ischemic bowel; Eye:such as cataractogenesis, retinopathy, degenerative retinal damage;Vessels: such as atherosclerosis and vasculitis; Erythrocytes: such asFanconi anemia, malaria; Heart: such as coronary thrombosis; Lung: suchas asthma, COPD; Kidney: such as transplantation, glomerulonephritis;Multiorgan: such as inflammation, cancer, ischemia-reflow states, drugtoxicity, iron overload, nutritional deficiencies, alcohol toxicity,radiation, ageing, amyloid diseases and toxic shock. The literaturerelated to the involvement of ROS in some diseases is the following:

Skin: Burn Youn, 1992 Solar Radiation Golan, 1994 Psoriasis Lange, 1998a, b Dermatitis Polla, 1992 Brain: Trauma Juurlink, 1998 Stroke ElKossi, 2000 Parkinson Ebadi, 1996 Neurotoxins Foler, 2000 AlzheimerSmith 2000 Joints: Rheumatoid arthritis Cimen, 2000 Gastrointestinaltract: Diabetes Gerber, 2000 Pancreatitis Sakorafas, 2000 Endotoxinliver injury McGuire, 1996 Ischemic bowel Lai, 2000 Eye:Cataractogenesis Eaton, 1994 Retinopathy of prematurity Hardy, 2000Degenerative retinal damage Castagne, 2000 Vessels: AtherosclerosisSingh, 1997 Erythrocytes: Anemia Anastassopoulou, 2000 Malaria Ginsburg,1999 Heart: Coronary thrombosis Chen, 1995 Lung: Asthma Montuschi, 1999COPD Montuschi, 2000 Kidney: Glomerulonephritis: Barros, 2001Multiorgan: Transplantation Jonas, 2000 Inflammation El-Kadi, 2000Cancer Prior, 2000 Ischemia Lewen, 2000 Drug toxicity Sinha, 1990 Ironoverload Karbownik, 2000 Nutritional deficiencies Olszewski, 1993Alcohol toxicity Lieber, 1997 Ageing Cadenas, 2000 Radiation Bednarska,2000 Amyloid diseases Floyd, 1999.

Besides therapeutic applications, it has been found that the thylakoidsof this invention may advantageously replace chloroplasts-derivedcompositions of the art that have been tested as biosensors orbiofilters or bioreactors. The art in the field teaches these specificuses, but the chloroplasts-derived compositions lack stability anddegrade very rapidly, which renders these uses unpractical from acommercial point of view. Therefore, a stable and dynamic thylakoidextract could advantageously substitute for these non-performingchloroplasts-derived compositions.

Biosensors:

Detection of toxic products is valuable for evaluating environmentalrisks associated with the presence of contaminants. Valid bioassayswould normally involve living organisms and would fulfil the followingminimal conditions:

-   i) they should be representative of the natural environment,-   ii) they should reproducible,-   iii) they should be reliable so as to provide no or almost no false    results; and-   iv) they should be sensitive.

Toxicity detection should also provide enough flexibility for analyzingdifferent types of contaminated samples. Toxicity should be ideallymonitored and sensed in real time fashion. Toxicity detection findsapplication in at least three industrial sectors: paper industry,contaminated soil analysis and agriculture. In all these instances,information is needed on the presence of contaminant in order to rapidlycorrect an undesirable situation.

A major problem encountered with the actual technologies to sense toxicproducts is in the long delay of obtention of the results of biotestsfrom 48 to 96 hours, when using organisms like trout or Daphnea magna. Agood detecting device would be one distinguishing from the availableconventional biotests by the use of material which would allowmeasurements of a contaminating potential of an effluent in real timeand continuously. Although some biodetectors are commercially sold,which measure fluorescence generated by plant photosynthetic activity, asystem that would permit measurement of electrical charges induced bythe presence of light, and modulated by the presence of contaminantswould be ideal. This technology would be much cheaper than thefluorimetric technology. It is believed that a technique which wouldevaluate the photosynthetic activity on a total thylakoid material wouldbe preferable over fluorometric methods which measure the activity of aspecific proteic complex, namely the PSII. A device comprising thylakoidmaterial would therefore have the advantage of measuring the toxicity ina larger spectrum of action. Such as detecting device would measure thenumber of electrons produced with a given light intensity. A current(Ep_(max)) obtained after a few seconds should be proportional to thephotosynthetic activity of the thylakoids. If the photocurrent isplotted against the concentration of contaminants, a typical sigmoidalshould be obtained, upon which an estimated EC₅₀ should be deduced.

A photocurrent has been already measured by Allen and Crane in 1976. Ithas been found that electron transport constituted a reliable andrepresentative measure of global photosynthetic activity and of thephysiological health of a plant. From the work of Allen and Crane, it isconceivable that an extract that would have a great stability, alongwith a dynamism and capacity to regenerate its responsive state tocontaminants and light would be highly preferable over the knowndevices. A detector would measure the number of electrons produced at agiven light intensity. A maximal photo-current value (Ip_(max)) obtainedafter a few seconds is proportional to the photosynthetic activity ofthe thylakoid membranes. If one plots Ip_(max) v. the concentration of aphotosynthetic inhibitor (a contaminant or a pollutant) present in thephotoconversion chamber, a typical sigmoidal curve is obtained. Theinhibitor potency can be easily evaluated (IC₅₀).

A detecting device would comprise: a white light source, aphotoconversion chamber receiving two electrodes, a detecting means formeasuring electrical currents induced by light and computer means forcollecting and processing data (electric currents). A liquid samplecomprising a toxic agent, a contaminant or a pollutant to be identifiedor measured, is contacted with a thylakoid membrane extract. Once themixture introduced in the chamber, a brief illumination is applied (lessthan one minute). The device or apparatus may be conceived to processand analyze a plurality of samples simultaneously.

Biofilters/Bioreactors:

Because the photosynthetic apparatus in plants is capable of not onlycapturing photons, but also of capturing and accumulating moleculeshaving affinity for its components, it is contemplated that the presentextract would also have the same capacity as the plant itself. Moreover,since some of the captured molecules may be processed, the presentextract would act as a bioreactor. The molecules susceptible to becaptured are, for example, herbicides, insecticides, fungicides, urea,ions and heavy metals as well as gas like O₃, CO, H₂S, NO, CO₂, O₂ . . .The biofilter of this invention would be versatible and would beresistant to temperature variations.

There is no existing practical process in the art teaching how torecover intact functional thylakoids, capable of retaining activity fora practical amount of time.

It is obvious from the above that the plants have a great naturalcapacity to manage with threatening situations. The thylakoids areparticularly adapted to resist and adapt to such extreme situations.

The U.S. Pat. No. 4,698,360 describes a plant extract comprisingpro-anthocyanidins useful as free-radical scavengers. The process ofmaking this extract comprises the following steps:

-   a) the obtention of a coarse powder of maritime pine bark;-   b) its extraction in boiling water;-   c) a separation of liquids from solids;-   d) cooling the liquids to ambient temperature;-   e) a filtration;-   f) a “salting-out” precipitation to remove undesirable matter;-   g) extracting active ingredients into ethyl acetate;-   h) drying the organic phase;-   i) resuspending the solids and reprecipitating the active    ingredients with chloroform; and-   j) resuspending the solids before advanced purification.

This reference is concerned with the isolation of a specific type ofactive ingredient, and not with the preparation of thylakoids that wouldcontain a major portion of its photosynthetic components, in other wordswherein pigments would not be separated from each another.

This reference is indeed typical of the overall teachings in the generalart which the present invention pertains to. The prior art relatessystematically to the isolation of one or more given plant components,and not to the isolation of intact thylakoids comprising a major portionof their constituents preserved in an integral and functional state.

Glick et al. (1985) in Planta 164: 487–494 describe the variations instoichiometric ratios of photosystems II and I (PSII/PSI) when peas aresubmitted to different types of light. The electron transport capacityof PSI and PSII in the presence of indicators such as2,5-dimethyl-p-benzoquinone and NADP, which are indicators specific forPSII and PSI, respectively. Although green light is used, which is anon-activating light environment, it is not used to condition the plantin a process which aims at isolating intact and activable thylakoids.The reference essentially relates to the study of the composition ofchloroplasts and not the preservation of thylakoids activity in functionof a given light quality and intensity. The plants are ratherconditioned in different lights that are depleted or enriched in redwavelengths. This reference is not concerned with the fact that thephotosynthetic pigments should be kept close to each other so as tofavorise the energy transfer between chlorophylls and carotenoids and tofavorise free-radicals capture. Thus the conditions leading to theisolation of photosynthetic pigments in their natural state inthylakoids are not specifically taught and met with in this reference.

Mason et al. (1991) in Plant Physiol. 97: 1576–1580 teach a method forisolating chloroplasts, which makes use of a step of forced passage of aplant suspension through a 27-needle at a flow speed of 0.5 ml persecond, rather than using a dispersion step by homogenization. The plantsolution comprises a buffer having a pH 7.5 and comprising 0.3 Msorbitol. The preparation that has been forced through the needle iscentrifuged in a Percoll gradient and the chloroplasts are separatedfrom other constituents, including thylakoids. This process is thereforedifferent from the present process which aims essentially at therecovery of thylakoids using quite simple steps and reactants, whichpresent process being also easy to scale up. The light conditions arenot mentioned in this reference. Further, the conditions to keepchloroplasts integral are obviously not the same as conditions todisintegrate chloroplasts. In the present process, the chloroplasts aredisintegrated but thylakoids membranes are recovered substantiallyintact. This reference therefore cannot teach the present invention.

The Canadian patent application 2,110,038 describes a process ofstabilizing plant extracts. These extracts are however cell fluids orjuices and not thylakoid membranes. There is no mention in thisreference of withdrawal of water as a natural electron donor from themembranes, for the purpose of stabilizing thylakoids.

In view of the foregoing, no practical process has been taught in theart, that would lead to the isolation of intact and functional thylakoidmembranes. There is further no teaching of conditions for stabilizingthylakoid components. There is finally no teaching of the use ofisolated thylakoid membranes to scavenge cell components from ROS.

There is therefore an open challenge in developing a process forobtaining active thylakoids that remain integral and, optionallyactivable, for an acceptable amount of time and which, upon reactivationare capable of acting as an antioxidant by their ROS scavengingactivity. Although an increasing body of literature is available onphotosystem components, nobody has published a practical process whereinthe conditions of isolation and preservation of thylakoid activity aretaught.

Moreover, since free radicals may be responsible for the degradation ofmany cell components, it is expected that their capture would protectother plant constituents. The present process would therefore produce animproved yield of plant components other than thylakoids.

Because there is a demand for powerful antioxidants, particularly in thepharmaceutical field, a process providing any such antioxidants, as wellas the antioxidants per se capable of a good potency as well as of asustained activity, would be greatly appreciated. Further, there is ademand for biological material useful as sensors or detectors, captorsor filters, bioreactors or biological molecule producers.

SUMMARY OF THE INVENTION

The present invention aims at providing a simple process for obtainingan extract having functional thylakoids. The present invention alsoprovides a process wherein the thylakoids are purified from other cellcomponents. It is another object of this invention to provide astabilized extract comprising non-isolated or isolated thylakoids. Thestabilized extract is essentially free of any electron donor which wouldactivate the thylakoids. Since the most abundant electron donor iswater, the stabilized extract is therefore preferably water-free. Watercan be chased by a solvent or by drying, for example. An amphotericsolvent, particularly a surfactant such as propylene glycol has beentried with success. This type of solvent does not disintegrate themembrane structural components, and has the advantage of replacing watermolecules and of preventing the formation of aggregates uponredissolution in an aqueous solution. The stabilized extract has alonger shelf life with no substantial loss of activity as long as noelectron donor such as water is added thereto. The stabilized extract isrehydrated extemporaneously before use to start the activation. Theactivity of extract once activated, lasts much longer than any otherknown antioxidant, which indicates a certain level of regeneration ofactivity rather than immediate and complete exhaustion. Further theantioxidant potency adapts, thus increases or decreases, upon the extentof the oxidative insult.

In accordance with the present invention, is provided a method asdefined in claim 1.

This invention provides a method of obtaining an extract obtainable fromphotosynthetic organisms comprising thylakoids, the method comprisingthe steps of:

-   a) providing a suspension of organism constituents that contains    thylakoids; and-   b) disrupting the constituents while maintaining thylakoids intact    in a medium having a viscosity comprised between 1 to 1.3 centipoise    and pH above 2 and below 10; the medium being added in a volume    calculated upon the following equation:    (Volume of medium+plant constituents water content)>10

Plant constituents dry weight whereby a first extract essentiallyconstituted of thylakoids, cells debris/membranes and a liquid phase isobtained, said thylakoids comprising integral photosynthetic pigments.

Preferably, the resultant of the above equation would be comprisedbetween 25 and 150.

The pH of the medium is preferably comprised between 5 and 8, morepreferably between 7 and 7.5.

The suspension of step a) may be obtained by mechanically dispersingorganism constituents or tissues in said medium.

In a preferred embodiment, step a) is preceded by a step of submittingsaid organism to a conditioning parameter selected from light, osmoticstress, heat, cold, freezing, dryness, hormones, chemical and biologicalinducers.

In a most preferred embodiment, step a) is preceded by a step ofconditioning said organism in a light environment of a wavelengthcomprised between about 500 and 600 nm, and step b) is performed underthe same light environment.

The viscosity is partly achieved by adding a sugar. The sugar may beadded in concentration as high as 1.5 M and over. Preferably, a sucroseconcentration of about 0.2 to 0.4 M in said solution or a sugarachieving a viscosity equivalent to 0.2 to 0.4 M sucrose.

A specific example of a medium used in the above method is: Tris oracetate or ascorbate buffer (20 mM, pH 7.0–7.5), and sorbitol or sucrose350 mM.

The method of this invention may further comprise the following step c):separating thylakoids, cell debris/membranes and liquid phase from eachanother, to form a second, third and fourth extracts essentiallyconstituted by isolated thylakoids, cell debris and membranes, andliquid phase, respectively.

The step of separating has been particularly performed upon a differenceof sedimentation coefficient of each of thylakoids, cell debris andmembranes, and liquid phase.

A specific example of such separating step comprises centrifuging thefirst extract for 10 minutes at 10 000 g in a tube equipped with afilter in a superior portion of the tube, the filter having a suitableporosity onto which cell debris and membranes deposit while thethylakoids and the liquid phase pass through the filter, the thylakoidsforming a pellet in an inferior portion of the tube. Alternatively,gross purification may be achieved by recovering cell debris andmembranes first by pressing and/or filtering, for example, followed by afiner purification, e.g. separating thylakoids from the liquid phase.

After separation, each first to four extracts may be stabilized byadding the following step d): eliminating any electron donor from saidextracts so as to inactivate and stabilize the photosynthetic pigmentspreferably in the presence of sugars (which may protect componentsagainst cold). The second and third extracts are particularly targetedby this step.

The first contemplated electron donor is water, so the extracts areprocessed to be water free.

Water may be eliminated under vacuum freeze drying or by exchanging itagainst a non-denaturing amphoteric solvent or surfactant after step c),non-denaturing meaning not capable of dissociating or of damaging thethylakoid structural components.

An amphoteric solvent which has been tried with success is propyleneglycol.

It is further another object of this invention to provide products thatresult from the above process. A pure thylakoid extract having thecapacity to be activable is first provided. A stabilized extract ispreferred. The above third extract being rich in thylakoids andcellulosic material is also within the scope of this invention, namelyin a stabilized form. The stabilized form for thylakoids may be dried orin a medium composed of an amphoteric solvent such as propylene glycol.The former is in an insoluble state or suspension; the latter forms asolution. Thylakoids comprising extracts are reactivated in the presenceof an electron donor. The first contemplated electron donor is water.Once activated, the extracts act as dynamic scavengers of ROS.

For nutraceutical, cosmeceutical and pharmaceutical applications, thisscavenging activity results in the treatment or the prevention ofdiseases or disorders that are mediated by the formation of ROS,especially those having an etiology related to inflammation, cancer, orcontact with radiations.

A first scavenging and protecting effect is exploited against radiationsthat are in the ultraviolet spectrum. Therefore topical use for thethylakoids and topical compositions comprising the thylakoids are withinthe scope of this invention.

It has been found that thylakoids have the capacity to form aphoton-absorbing film or coating on a body surface like skin or mucosa.This property appears to be independent from the ROS scavengingactivity. The extracts comprising thylakoids, in an activable form ornot, therefore act as a filter for radiations, namely in the ultravioletrange. When the extracts further have functional thylakoids, they have adual role as U.V. filter and as a ROS scavenging compound. The extractsmay be used further in a method or a composition or a device fordetecting, for capturing molecules or for producing or processingmolecules having affinity for thylakoids or interfering with theiractivity.

Examples of such molecules are herbicides such as triazines (ex.atrazine- and diuron-type herbicides), quinones, chlorpromazine, urea,formaldehyde, alkylamino cyanoacrylates, trypsin, cyanoacrylate, Tris,adenine derivatives, disulfiran (metal chelator), acetyl CoAcarboxylase, digitonin, heavy metals (ex., Cu, Zn, Cd, Pb, Hg . . . ),SO₂, NO₂, NH₂OH, CO₂, CO, O₃, O₂, H₂S, calcium antagonists(calmodulin-type), sulfate, sulfite, bisulfite, nitrite, acetate,lactate, anions such as NO₃ ⁻, HCO₃ ⁻, HCO₂ ⁻, F⁻, NO₂ ⁻, HS0₃ ⁻, . . ..

DESCRIPTION OF THE INVENTION

This invention will be described hereinbelow, referring to specificembodiments and the appended figures, the purpose thereof being toillustrate this invention rather than to limit its scope.

DESCRIPTION OF THE INVENTION

This invention will be described hereinbelow, referring to specificembodiments and the appended figures, the purpose thereof being toillustrate this invention rather than to limit its scope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the relative antioxidant activity of the extract ofthe present invention in function of the pH of the exogenous extractionfluid.

FIG. 2 shows the relative activity of the extract of the presentinvention in function of the sorbitol concentration included in theextraction fluid.

FIG. 3 shows the relative activity of the extract of the presentinvention in function of time and of the sugar concentration in theextraction fluid. Each sample was kept at −20° C.

FIG. 4 represents the relative activity of the extract of the presentinvention in function of the nature of the sugar in the extractionfluid.

FIG. 5 shows the relative activity of the extract of the presentinvention in function of different salts used for extraction.

FIG. 6 represents the relative activity of the extract of the presentinvention (FRTS non-stabilized) in function of time and temperature.

FIG. 7 shows the selection of centrifuge conditions for optimization.

FIG. 8 shows the relative activity of the extract of the presentinvention in function of the proportion of propylene glycol (propane 1,2diol) included in the resuspension solution.

FIG. 9 shows oxidation curves. Curve A represents a lipid oxidationwithout any antioxidant; curve B shows lipid oxidation in the presenceof an anti-oxidant which “allows” some lipid oxidation; curve Crepresents lipid oxidation in the presence of an efficient radical chainbreaking antioxidant, such as vitamin E.

FIG. 10 illustrates the oxidation kinetics of lipids PLPC, wherein Bstands for lipids without the extract of this invention, D stands forlipids in the presence of Trolox, L stands for lipids in the presence ofan active extract made in accordance with this invention and T standsfor lipids in the presence of lipids treated with an inactive extract.

FIG. 11 illustrates the protection exerted by the extract of the presentinvention (dilution 1:1000) on IMR-32 cells against damages caused bytwo concentrations of TBHP; FIG. 11 a): 25 μM; FIG. 11 b): 50 μM.

FIG. 12 represents the protection exerted by two different dilutions ofthe extract of the present invention on IMR-32 cells after TBHPtreatment; FIG. 12 a): dilution 1:1000; FIG. 12 b): dilution 1:10000.

FIG. 13 illustrates the behaviour of brain hyppocampus slices to a 120sec. epoxia and recovery in the presence of a solution comprising theextract of the present invention or not (control).

As used herein, an “antioxidant” is a substance that, when present in amixture or structure containing an oxidizable biological substrate,significantly delays or prevents oxidation of the biological substrate.Antioxidants can act by scavenging biologically important reactive freeradicals or other ROS (singlet oxygen, .O2-, H₂O₂, .OH, HOCl ferryl,peroxyl, peroxynitrite, alkoxyl . . . ), or by preventing theirformation, or by catalytically converting the free radical or other ROSto a less reactive species.

The antioxidant of the present invention is considered as such if, whenadded to a cell culture or assay reaction, it produces a detectabledecrease in the amount of a free radical, such as superoxide, or anonradical ROS, such as hydrogen peroxide or singlet oxygen, as comparedto a parallel cell culture or assay reaction that is not treated withthe antioxidant. Suitable concentrations (i.e., efficacious doses) canbe determined by various methods, including generating an empiricaldose-response curve, predicting potency and efficacy of a congener byusing QSAR methods or molecular modeling, and other methods used in thepharmaceutical sciences.

The present invention is intended to be used in the medical field totreat, prevent, or alleviate the symptoms associated with a ROS,associated disease or disorder or reduce the expression of such diseaseor disorder. Such a disease or disorder refers to a condition of anindividual that results at least in part from the production of orexposure to free radicals, particularly oxyradicals, and other “ROS” invivo. Even though there is only a few if any pathological conditionsthat are monofactorial, there is an increasing body of literature andknowledge related to the involvement of ROS in disease etiology. Forthese reasons, the term “ROS associated disease” encompassespathological states that are recognized in the art as being conditionswherein damage from ROS is believed to contribute to the pathology ofthe disease state, or wherein administration of a free radical inhibitor(e.g., desferrioxamine), scavenger (e.g., tocopherol, glutathione), orcatalyst (e.g., SOD. catalase) is shown to produce a detectable benefitby decreasing symptoms. increasing survival, or providing otherdetectable clinical benefits in treating or preventing the pathologicalstate. For example but not limitation, the disease states discussedherein are considered ROS-associated diseases (e.g., ischemicreperfusion injury, inflammatory diseases, systemic lupus erythematosis,myocardial infarction, stroke, traumatic hemorrhage, spinal cord trauma,Crohn's disease, autoimmune diseases (e.g., rheumatoid arthritis,diabetes), cataract formation, uveitis, emphysema, gastric ulcers,oxygen toxicity, neoplasia, undesired cell apoptosis, radiationsickness. Further, many inflammatory diseases or disorders will benefitof the present invention, since it is known that ROS intervene in theprocess of inflammation. For example, the “oxidative burst” of activatedneutrophils produces abundant superoxide radical, which is believed tobe an essential factor in producting the cytotoxic effect of activedneutrophils. Further, since neutrophils are involved in the earlymortality of any grafted or transplanted tissue or cell, an antioxidantwould increase the early survival of transplanted or grafted cells,which is critical for the success of transplantation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to isolated thylakoids and a method forisolation of thylakoids, that will constitute a powerful antioxidantmolecule having a scavenger activity towards ROS. This antioxidant is ofa natural origin; it should have no toxicity or adverse effect, whenemployed in a reasonable concentration. This antioxidant can also bestabilized, which ensures stability over time, thus a reasonableshelf-life. Stabilization is performed by withdrawing electron donors(namely water molecules), which make thylakoids to stay in a quiescentform. Thylakoids are activated by adding an electron donor (namelythrough hydration).

Preparation or Conditioning:

A first step undertaken, before going through the steps for recoveringthe thylakoids in a crude suspension, may be a conditioning step. Thisconditioning is optional and permits to vary the compositions of theextracts. To optimize the levels of pigments in their non-activatedstate (namely chlorophyll and carotenoids), a conditioning step may beperformed in the same conditions as the working conditions, e.g. undergreen light or in the dark. Under such circumstances, the chlorophyllsare preferably in a singlet state while the carotenoids are preferablyin a fundamental state. This way, when ready to use, the carotenoidswill be activated and ready to take the energy coming from a tripletchlorophyll (photoprotection).

It is also possible to further protect the thylakoid pigments by addingother xanthophylls such as violaxanthin in the medium of extraction orto increase the number of carotenoids by working under a light having anarrow range of wavelengths (465–475 nm).

It is further also possible to enrich the organism, namely a plant, andits extracts, in some particular constituents by submitting the organismto a conditioning step other than light conditioning. Such otherconditioning comprises osmotic stress, heat, cold, freezing, dryness,hormones, and chemical and biological inducers. All these conditioningparameters lead to a response in sensitive organisms, which then becomeenriched in said some particular constituents.

As an example of this, a heat treatment would promote the accumulationof heat shock proteins, that are useful for treating ROS-relateddiseases or disorders (namely arthritis). The main objective of thesteps of the present process is to preserve the integrity of somevaluable constituents, namely the molecular constituents of thylakoids,and to control the state of the molecules, preferably in theirfundamental functional state.

Obtention of a Crude Extract:

When one starts with whole organisms or tissues thereof, such as planttissues or whole plants, the first step of the process is a dispersingstep such as a homogenization step. The plant tissues are, for example,pulverized mechanically. The mesophylium tissues (leaves or needles) maybe cut into small pieces with the aid of a rotative knife such as thatretrieved in a homogenizer or a commercial rotative cutter. Any meansleading to the dissociation of the cellulosic material to uncover thethylakoids would be suitable.

Besides working under a light source which optimally minimize the lightflux (green light, λ=500–600 nm), the working conditions would ideallycomprise a working temperature of about 2 to 20° C., preferably lessthan 4° C., for the purpose of increasing the cell density and ofpreventing any degradation by enzymes. The working conditions alsoinclude hypertonic conditions using hypertonic agents such as sugars.These conditions achieve optimal viscosity and fluidity. A specificexample of a homogenization buffer is as follows:

Homogenization Medium

Volume, Final weight Product pH Molarity    6 ml Tris Buffer (1 M) 7.0 20 mM   50 ml Sorbitol (2 M) 330 mM  1.5 ml MgCl₂ (1 M)  5 mM 243.5 mlH₂O   300 ml Total

The pH of the solution can vary from above 2 to below 10 preferably from5 to 8, more preferably maintained at a near neutral value of 7–7.5(FIG. 1).

Taking spinach as a reference plant, the ratio wet weight of plant leaftissues (g)/volume of buffer (ml) is of about ⅓. Thus, the above recipeis suitable for extracting thylakoids from 100 g of spinach. The plantis mixed with the buffer and homogenized for example, in a domesticblender for about 30 seconds. The plant source may vary, so does themedium volume. The buffer itself may be any one suitable for maintaininga near neutral pH. For example, the above Tris buffer may be replacedwith an acetate or ascorbate buffer. Both substitute buffers areacceptable for human consumption and ascorbic further has the advantageof providing vitamin C to the consumer. The sorbitol has been added topreserve the integrity of the membrane (FIGS. 2 and 3) and to insure aviscosity varying from about 1 to 1.3 and may be replaced by any othersuitable sugar such as commercial saccharose, fructose or turbinado in aconcentration achieving the same effect as 0.2 to 1.5 M (preferably0.2–0.4 M) sorbitol. Sucrose 0.2–0.4 M would be an acceptable lessexpensive component (FIG. 4). Buffer components such as MgCl₂, NaCl,ascorbic salt/acid are not believed to be necessary to the presentprocess, but they may help recovery more activity or preserving theactivity for a long period (FIG. 5).

A near neutral pH was preferably selected for maintaining an optimalconcentration of H⁺ions. Sugars and pH are important parameters forpreventing the dissociation of photosynthetic pigments. The density ofcell fluids is maximized when working in a cool or cold environment,namely below 4° C. (see FIG. 6, wherein FRTS/1 stands for the presentextract). Low temperatures also may protect components from enzymaticdegradation. All these homogenization conditions release the membranestructure from its organization in chloroplasts without substantiallyaffecting the molecular structural organization of thylakoids. Thechloroplasts are therefore disorganized without destroying ordisintegrating the thylakoids. The surface of cell components withoutany cellulosic protection is thus increased.

It was convenient in the present process to use plant tissues directlyin an extraction medium. However, if it becomes advantageous to use purechloroplasts or a preparation enriched in chloroplasts or evenpreparation of other photosynthetic organisms having or notchloroplasts, it is feasible to do so. Cultured cells or tissues canalso obviously replace whole plants.

Starting from spinach leaves, the yield of thylakoids is fairly goodwhen one follows the following equation:

α/β > 10${\alpha = {{ratio}\mspace{14mu}{of}\mspace{14mu}{wet}\mspace{14mu}{weight}\text{/}{dry}\mspace{14mu}{weight}}};{{{and}\mspace{14mu}\beta} = {{ratio}\mspace{14mu}{of}\mspace{20mu}{wet}\mspace{14mu}{{weight}/{\left( {{{Volume}\mspace{14mu}{of}\mspace{14mu}{medium}} + {{plant}\mspace{14mu}{constituents}\mspace{14mu}{water}\mspace{14mu}{content}}} \right).\text{}{{So}:{\frac{\left( {{{Volume}\mspace{14mu}{of}\mspace{14mu}{medium}} + {{plant}\mspace{14mu}{constituents}\mspace{14mu}{water}\mspace{14mu}{content}}} \right)}{{Plant}\mspace{20mu}{constituents}\mspace{14mu}{dry}\mspace{20mu}{weight}} > 10}}}}}}$And  more  precisely(,  preferably)$\frac{\left( {{{Volume}\mspace{14mu}{of}\mspace{14mu}{medium}} + {{plant}\mspace{20mu}{constituents}\mspace{20mu}{water}\mspace{14mu}{content}}} \right)}{{Plant}\mspace{20mu}{constituents}\mspace{20mu}{dry}\mspace{14mu}{weight}} = {25 - 150}$

It is worthwhile noting that the yield may vary depending on the volumeof buffer that was selected and on the water content of the selectedplant. For example, pine needles have an endogenous water content thatis much less important than in the case of spinach leaves. For an equalwet weight of plant material the volume of buffer should be increasedfor isolating thylakoids from pine needles, when compared to the spinachleaves, taking into account all the parameters of the above equation.

The crude extract obtained alone constitutes a first fraction that canbe used per se, dehydrated, or further fractionnated.

Separation of Plant Fractions:

The homogenization step is followed by a separation step. Thylakoids areseparated from cell debris and from soluble components, based on theirdifferent sedimentation coefficients. The sedimentation coefficient ofthylakoids is superior to that of cell organelles. The thylakoids werecentrifuged for 10 minutes at 10,000×g in mobile buckets. A centrifugeforce of less than 10,000×g but superior to 3,000 g may be used,adjusting the centrifugation time accordingly (FIG. 7). The optimalhandiness for the thylakoids pellets was obtained at 10000–12000×g for10 minutes. Any other speed and time achieving equivalent results may beadopted. Different speed and time are contemplated in a scaling upprocess. During sedimentation, the thylakoids pass through a filtercorresponding to this equation:0.002≦X≦0.2

wherein X is calculated by multiplying the opening per the wire diameter(all in millimeters). The cell debris and membranes are stopped by thisfilter in a superior portion of a centrifugation tube. Thus, the bottompellets comprising the thylakoids are easily recovered and a pellet maybe used immediately or may be further fractionated or stabilized for anyfuture use. Of course, any other method of separation achieving the samepurpose of isolating thylakoids could be used. For example, on a densitygradient like a sucrose gradient could be used. Any chromatographic oraffinity medium and method could be also used. Referring to the abovespecific method, it is conceivable that the gross and fine separationwould not be achieved in one step in a large scale process. Therefore, agross purification could be made first on a press or a filter and thefine separation of thylakoids and the liquid phase would be achieved ina later step.

Stabilization:

The separation step is normally followed by a stabilization step. Thisstep allows withdrawal of electron donors such as water molecules thatare bound or non-bound to membranes, and this for eliminating theactivator of the PSII system. The fractions are recovered and are placedin clean vials. Specifically the first, second and third fractions,representing the whole extract, the pellet (thylakoid fraction) and thecell debris/membranes fractions, respectively, are lyophilized. Thevials are then submitted to a vacuum and to a low temperature (about −20to −50° C.), during at least 4 hours. The fractions so lyophilizedremain stable for a long time, until water is added thereto. Otherstabilizing means could be used. For example, a plurality of surfactantshave been used to verify their capacity to chase water withoutdestroying the structure of the thylakoids. These solvents are thefollowing: Triton X-100, PEG, Beta-D-maltoside, glycine, glycerine,glycerol, TWEENs™, SDS, LDS, DMSO, cholate, stearate and propyleneglycol have been used.

Propylene glycol has been preferred and would advantageously replacelyophilization as a stabilizer. Not only propylene glycol provides aninactive quiescent thylakoid preparation (functional and fully activableupon water addition, see FIG. 8), but it also stabilize the thylakoidsupon hydration by helping solubilizing the same. Upon hydration,thylakoids normally form a suspension; in the presence of 100% propyleneglycol, thylakoids form a solution having of limit of solubility of 100mg/mL solution. This solution may be diluted with water for activation.This solvent is also non toxic.

Optional Fractionation:

The thylakoid membranes could further be fractionated intosub-fractions. For example, it could be envisaged to separate reactionalcenters or photosystems, light harvesting complexes, cytochromecomplexes, particular pigments (chlorophylls, carotenoids),plastoquinones, non-photosynthetic, components (cell nuclei), ormitochondria.

Thylakoid Integrity and Activity:

The extract comprises substantially pure thylakoids (>90%); they arephotosynthetically activable; they are stable; and the extract iscontrollable. The photosynthetic activity has been evaluated withdifferent techniques: the oxygen release (Schlodder et al. 1999), thephotoreduction of 2,6 dichlorophenol indophenol (DCPIP) (Behera et al.1983) and the fluorescence (Maxwell et al. 2000). Moreover, theintegrity of the thylakoids has been evaluated with a technique whichmeasures a continuous electric current: any disorganization should bedetected by any variation in this electric current. The current ismeasured from PSII to the coupling factor, which indicates that thethylakoids contain the main subunits listed above and that they arefunctional.

When a green light was used in the working conditions, the pigments werestabilized in their fundamental state (F₀), thus, permitting theoptimization and synchronization of any desired effect. Thestabilization is possible because of the withdrawal of the primaryelectron donor. The stability measured by the photosynthetic activity(absent during quiescent state and present upon activation with anelectron donor) and the concentration in chlorophylls and carotenoids,persist for several months after extraction. The ratiochlorophylls/carotenoids is also extremely important for the activity ofthe complex and to maximize the absorption and dissipation of energy.

The extracts are easily detectable because of their naturalfluorescence. No toxic product, solvent, detergent or conservation agenthas been added to the above thylakoids, preserving to the product allits original nature. The extracts are indeed edible. Even when propyleneglycol is used to stabilize the thylakoids, this solvent is harmlessbecause its oxidation yields pyruvic and acetic acids. This solvent iscurrently used as a food emulsifier, which means that it has surfactantproperties (however, non deleterious to the integrity of thethylakoids). It further has an inhibitory activity against fermentationand mold growth. Therefore, this solvent may be used at any step in theprocess, mixed with water during homogenization, and not mixed withwater after step c) (separation step).

The extracts may be presented under a solid form, dry or humid, or in aliquid form. The extracts are poorly soluble in water although theyrehydrate easily but they resuspend completely in propylene glycol.Thylakoids are reactivated upon rehydration. It is thereforeenvisageable that a composition comprising solubilized thylakoids isused; when contacted with an aqueous medium, the thylakoids activate.

Byproducts:

Although the thylakoids are the products that have received the primaryattention in the above procedure, the other plant constituents that areseparated from the thylakoids will also be recovered for theircommercial value. The liquid phase fraction and the cell debris/membranefraction may be easily taken as starting material to isolate any plantmolecule of interest. The latter fraction may be reextracted to increasethe yield in thylakoids that are recovered per plant unit. It iscontemplated that the components of other fractions would have asuperior quality when compared to any corresponding components obtainedfrom the processes of the prior art. Because the formation of damagingROS is prevented or because already formed ROS are captured by thethylakoids prepared in accordance with the present process, it isenvisageable that any other plant constituents sensitive to ROS willalso benefit from the present process. Indeed, the other constituentsthat would be normally prone to degrade upon oxidation will be preservedby eliminating a noxious source of degradation. Thus, any plantconstituents such as sugars, proteins, lipids, vitamins, minerals andhormones can be separately obtained by fractionating, for example, theliquid phase obtained from the above process, which constituents wouldhave a greater specific activity than usual. In addition to this, aproper conditioning step may enrich the extracts in constituents ofinterest.

After verifying that the extracts were functional, the next step was toverify their scavenger activity towards ROS.

The Use of Thylakoids as Antioxidants:

Antioxidants are compounds that interact with ROS (such as oxygensinglet, hydrogen peroxide, superoxide anions and hydroxyl radicals). Inorder to form innocuous degradation products, active oxygen formsdegrade or inactivate other molecules and, potentially cause mutations,cancer or inflammation. They may further participate into aging. Theantioxidant molecules of the present extracts are the following:chlorophylls, carotenoids and vitamins (B, C, E, K, . . . ), cytochromesand anthocyanins. The thylakoids are particularly performingantioxidants because their physical organization makes the carotenoidscapable of capturing singlet oxygen in their chlorophyll protectiverole. The quenching effect has both a high efficacy and a relativelylong duration because the carotenoids dissipate their energy as heat andbecome ready to accept again the energy coming from tripletchlorophylls. The thylakoids are therefore outstanding in the field ofantioxidants; because of their capacity to regenerate their functionalstate, the organization of the pigments will permit a sustainedanti-oxidative activity.

The thylakoids of this invention will hereinbelow be referred to asFRTS/1 constitute a bioactive molecular complex extracted from plantbiomass. The functional anti-oxidative activity of FRTS/1 is based onthe redox potential of the molecular complexes of thylakoids wherein thetridimensional structure and the natural distance between its differentpigments and molecules is preserved. The antioxidant is an indication ofan optimal structure and of an optimal composition of matter.

Quantification of Proteins by Fluorescamine Method

The protein concentration in a stabilized extract is : 0.42–0.65 g/g ofextract.

Antioxidative Function of the Thylakoids

A—Principal Reaction Pathway in Radical-Induced Lipid Oxidation:

Nature employs antioxidants to prevent the oxidation of biomoleculessuch as proteins, DNA, lipids, etc. The peroxidation of lipids is aparticularly ubiquitous and damaging process in living organisms.Peroxyl radicals (ROO.) are important radicals in biological systemsbecause they are able to initiate lipid peroxidation and they areintermediates in many different oxidation processes of biologicalimportant molecules.

The peroxidation of unsaturated lipid moieties (reactions 1–3) causeschanges in their structure which eventually changes the physicalproperties of biological membranes. During the lipid peroxidation aconjugated diene group is formed (see LOOH in reaction 3) which has anUV/Vis absorption maximum at λ_(max)=234 nm (ε=29,500 M⁻¹ cm⁻¹) and thisallows a quantification of the oxidation product formed. To obtainquantitative data the lipid peroxidation has to be initiated in acontrolled manner (reaction 1). Often azo initiators, which produce awell-defined flux of ROO. in aqueous, aerated solution, are employed tostudy lipid oxidation in in vitro experiments. The most commonly usedwater-soluble azo initiator is AAPH (sometimes called ABAP; reaction 4).

The decomposition rate of AAPH at 37° C. is k=1.3×10⁻⁶ M⁻¹ s⁻¹ and theefficiency for ROO. formation is 50%, i.e. 1 mol AAPH yields 1 mol ofROO. This method of ROO. generation allows the calculation of the exactamount of ROO. formed during any time period.

The most intensively studied antioxidant is Vitamin E and the mostactive compound of the Vitamin E family is α-tocopherol (α-TocH). It isa radical chain breaking antioxidant which can trap two peroxyl radicalsyielding only non-radical products and thereby preventing lipidperoxidation (reactions 5 and 6).ROO.+TocH→ROOH+Toc.  (5)ROO.+Toc.→non-radical products  (6)

The tocopheroxyl radical (Toc.) formed in reaction 5 is a relativelyunreactive radical which normally cannot propagate the radical oxidationchain reaction. As soon as all TocH is consumed the lipid peroxidationoccurs as if no antioxidant is present.

Carotenoids (Car) are the most likely “antioxidants” in FRTS/1 becausechloroplasts utilize carotenoids such as carotenes and xanthophylls fortheir exciton transport chain. There are several pathways for reactionof carotenoids with ROS possible and the overall behavior of them rangesfrom antioxidant activity all the way to being effectiveless withrespect to lipid peroxidation inhibition. The experimental resultsobtained are depending on the reaction conditions employed. Possiblereactions of carotenoids with ROO. are:

Some reactions lead to the formation of non-radical products (reactions7+8, 10+11, 12+14) which would result in an overall antioxidant behaviorof a carotenoid. Other reactions are generating peroxyl radicals(reactions 9 and 13) which can be involved in the propagation of lipidperoxidation. The overall behavior of carotenoids cannot be obviouslypredicted due to the various possible reaction pathways.

Depending on possible reaction pathways of the antioxidant used theconcentration time profiles of the detected LOOH display certaincharacteristics. By using an azo initiator as peroxyl radical source theamount of ROO. generated can be calculated and it is possible todetermine the amount of lipids oxidized from the 234 nm absorption ofthe conjugated diene moiety. Also the amount of ROO. trapped during thelag phase can be determined.

A process within the FRTS/1 might “restore” is “original” antioxidativeproperties. A possible mechanism for such a behavior might be anelectron transfer rather than a radical trapping process (see reactions15 and 16), e.g.:

The concentration/time profiles of the lipid oxidation in the presenceof FRTS/1 will allow examining this hypothesis. From the duration of apossible lag phase during lipid peroxidation experiments (FIG. 9) theamount of “trapped” radicals can be calculated. This will allow to drawconclusion to which extend FRTS/1 is able to inactivate peroxylradicals. However, it has to be born in mind that the described possibleregeneration of the antioxidative properties can be only effective aslong as FRTS/1 is still intact and able to perform its “original”activity. Overall the experiments will eventually provide quantitativedata for the antioxidative capacity of FRTS/1 (see FIG. 10 for anexample of the antioxidative kinetics of FRTS/1 in comparison ofTrolox). When β-carotene was compared to Trolox, the former was anantioxidant but with no lag phase typical of antioxidants.

Since lipids are the main components of the cell membrane, lipoproteinsand other membrane structures in living organisms, in the present studyPLPC-FRTS/1 (1-palmmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine)micelles were used as a substrate for the standard oxidation assay.Oxidation of PLPC-FRTS/1 induced by peroxyl radicals generated from theinitiator Azo compound 2,2′ Azobis-(2-amidinopropane) dihydrochloride(AAPH) results in oxidation of the linoleic acid moiety to thecorresponding hydroperoxide together with the formation of a conjugateddiem system with an absorption maximum at 234 nm.

Preparation ofPLPC(I-palmmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine)Micelles:

170 μL of a 25 mg/mL solution of PLPC-FRTS/1 in CHCl₃ (Avanti PolarLipids) was evaporated to dryness under a stream of N₂. Phosphatebuffered saline (PBS) (281 μL) which had been previously treated withChelex® to remove metal ions was added to the PLPC-FRTS/1 and themixture was vortexed 2 min. Aqueous Chelex® treated sodium cholate 109μL, 30 mg/mL, (Aldrich Chemical Company Inc., Milwaukee, Wis., U.S.A)was added to the mixture and vortexed 2 min. The mixture was passed 20times through a polycarbonate membrane (pore size 100 nm) in order tohomogenize the size of the micelles.

FRTS/1 was dissolved in CHCl₃ (12 mg/mL) and I ml of this solution wasmixed with 6 mL of PLPC-FRTS/1 (25 mg/mL) to obtain a finalconcentration of PC-FRTS/1 of 6 mg/ml. Micelles were prepared asdescribed above. Azo initiator (AAPH) was used at a concentration of 5mg/mL in PBS.

Standardization:

Initial experiments were performed using FRTS/1 in a dehydrated form, tostandardize the concentration of micelles, azo initiators andwavelengths. Optimal absorbance was at 234 nm.

Two preliminary experiments were run with 100 μl micelle solutionprepared from PLPC-FRTS/1 and PLPC-FRTS/1+FRTS/1 in 3 mL of PBS with twodilutions (10 μl and 20 μl) of 5 mg/mL solution of azo-initiator for 10h at 37° C. at wavelength 234 nm on a Cary 3 UV-Visiblespectrophotometer from Varian. The background absorption due to thePC-FRTS/1 at 234 nm was too high under these reaction conditions.

Experiments:

Based on the results obtained from the above experiments, it was decidedto use further diluted solutions of the FRTS/1 (final concentrations ofFRTS/1 were 6.7 μg). As negative control, the FRTS/1 was incorporatedinto micelles made from 1,2-Dimyristoyl-sn-glycero-3-phosphatydylcholine(DMPC) a compound which is resistant to oxidation mediated by peroxylradicals derived from AAPH. Micelles were prepared from 15.937 mg ofDMPC-FRTS/1 (stock solution 25 mg/ml) in 0.6375 mL of PBS+1.053 mL ofPBS+0.408 mL of Sodium cholate+0.2435 mL of FRTS/1 solution in CHCl₃ (2mg/mL) as described above. The following experiments were thereforeconducted:

-   3 mL of PBS+100 μl of PLPC-FRTS/1 micelles+10 μl of azo-initiator-   3 mL of PBS+100 μl of PLPC-FRTS/1 micelles+20 μl of azo-initiator-   3 mL of PBS+100 μl of DMPC+FRTS/1+10 μl of azo-initiator-   3 mL of PBS+100 μl of DMPC+FRTS/1 20 μl of azo-initiator-   3 mL of PBS+10 μl of azo-initiator-   3 mL of PBS+20 μl of azo-initiator

The reactions were monitored on a spectrophotometer at 37° C. for 10 h.

Conclusion

These results show that the maximum OD (at 234 nm wavelength) ofPLPC-FRTS/1 micelles containing FRTS/1 at 0.3 mg/mL was 0.25 after 180min while OD of PLPC-FRTS/1 micelles without FRTS/1 was 3.2 after thesame period of time. The results indicate that the FRTS/1 undoubtedlydemonstrate antioxidative properties.

Antioxidant Properties of FRTS/1 Solution in Comparison of Vitamin E

Lower concentrations of antioxidants was used to compare theantioxidative properties of FRTS/1 and Trolox, a water soluble analog ofVitamin E (0.3 mg/mL in PBS). The following samples were run onspectrophotometer for 10 h at 37° C.

-   1. 3000 μL of PBS+10 μL of azo-initiator.-   2. 3000 μL of PBS+100 μL of PLPC micelles+10 μL of azo-initiator.-   3. 2998 μL of PBS+2 μL of antioxidant (0.5 mg/mL aqueous    solution)+100 μL of PLPC micelles+10 μL of azo-initiator-   4. 2995 μL of PBS+5 μL of antioxidant (0.5 mg/mL aqueous    solution)+100 μL of PLPC micelles+10 μL of azo-initiator-   5. 2990 μL of PBS+10 μL of antioxidant (0.5 mg/mL aqueous    solution)+100 μL of PLPC micelles+10 μL of azo-initiator-   6. 2980 μL of PBS+20 μL of antioxidant (0.5 mg/mL aqueous    solution)+100 μL of PLPC micelles+10 μL of azo-initiator-   7. 2990 μL of PBS+10 μL of Trolox+100 μL of PLPC micelles+10 μL of    azo-initiator-   8. 2980 μL of PBS+20 μL of Trolox+100 μL of PLPC micelles+10 μL of    azo-initiator-   9. 2970 μL of PBS+30 μL of Trolox+100 μL of PLPC micelles+10 μL of    azo-initiator

FIG. 10 shows a more sustained activity for FRTS/1 than for Trolox.

Protection Mechanism:

Radical Oxygen Species (ROS) readily interact with cellularmacromolecules and structures, resulting in membrane permeabilitychanges, activation of proteases and nucleases, and altered geneexpression (Yu, 1994; Schiaffonati and Tiberio, 1997). It is well knownthat these cellular changes induced by ROS lead to apoptotic cell death.We attempted:

-   To evaluate the antioxidative properties of FRTS/I; and-   To determine the action mode of FRTS/1 as an antioxidant.

IMR-32 cells constitute a good model for evaluating the antioxidantpotency of our extract. These cells are neuroblastoma cells that aresensitive to an oxidative stress which provokes apoptosis.

Experiment 1. Standardization of Experimental Conditions

Selection of Cell Culture

Human neuroblastoma cell line (IMR-32),which is known to respond tooxidative stress by apoptosis (Kim et al, 1999), was used as an in vitrocell model. IMR32 cells are particularly sensitive to ROS and othertoxicants because their p53 gene product is sequestered in thecytoplasm. The sequestration renders the p53 inactive although the geneis not mutated.

Selection of ROS Inducer

Tert-butyl hydroperoxide (TBHP) was used as an oxidative stress-inducingagent. TBHP does not have any neuron specificity in contrast tooxidative stress induced by MPTP in dopaminergic neurons. This willallow comparisons if studies relative to more generalized oxidativestress conditions (like the ones found in many neurodegenerativediseases) have to be performed on different cells phenotypes.

A. Determination of Optimal Dosage of TBHP to Induce Apoptosis on IMR-32Cells Culture

Experiments were done using 1000 IMR-32 cells per well, for I hour ofincubation. TBHP 50, 75 and 100 μM produced 78%, 87% and 87% apoptosis.TBHP 50 μM was selected to induce apoptosis in the other experiments.

B. Dosage of FRTS/1

Different dilutions were used on IMR-32 cells along with TBHP. A mothersolution 1:10 was constituted, starting from the lyophilized thylakoidfraction, in propylene glycol. Unless otherwise specified, the mentioneddilutions are dilutions of this mother solution. The protectionconferred by FRTS/1 was 28%, 35% and 75% at dilutions 1:10, 1:100 and1:1000, respectively. The latter dilution was adopted for furtherexperimentation.

1. Cell Culture

IMR-32 Cells were grown in MEM supplemented with 10% FBS at 37° C. in ahumidified atmosphere of 95% air and 5% CO₂. The cells were seeded at adensity of 1×10⁴ cells/T25 Falcon tissue culture flasks and subculturedtwice weekly. 48 h old cultures were used in all the experiments. 1000cells were plated per well in the first experiments, the number wasincreased to 3000 afterwards as indicated in the tables.

2. Oxidative Stress in vitro Protocol

1000 or 3000 cells/well/100 uL were seeded in 96 well (Linbro flatbottom) plates in all the wells except well No. 12 of all the rows.After 24 h the cells were washed 2 times with 250 ul of PBS (pH 7.2) and32 wells each were treated respectively with 100 and 200 uM solutions ofTBHP (70% aqueous solution from Sigma Chemical Company) for 1 h. After 1h the cells were gently washed 2 times with PBS before adding freshgrowth medium. After 24 h the cell survival was assayed by a sensitivefluorimetric assay based on DNA binding fluorescent dye Hoechst by thefollowing procedure. The test measures the total DNA of the population,a measurement which closely correlates with cell number. The medium wasaspirated by gentle suction. Cells were rinsed with 250 ul PBS. PBS wasaspirated with gentle suction. The rinse step was repeated. 100 uL lysisbuffer (0.02% SDS in 1×SSC) was added in every well except the ones forDNA standard and Blank in row 12. The plate was incubated at 37° C. for1 h with occasional swirling. 100 uL of 40 ug/mL of DNA was added to theDNA wells and 100 uL of 1×SSC buffer was added to the wells that weretreated as Blank. 100 uL of 40 ug/ml of Hoechst 33258 in 1×SSC bufferwas added to every well, and the plate was covered with Aluminum foil toprotect it from light. The plate was agitated gently for 5 minutes andfluorescence was read at excitation wavelength 355 nm and emissionwavelength 460 nm.

3. Calculations

CT untreated=100%: survival after TBHP: 42%, dead cells: 100−42=58%;population size after PC: 88% difference from CT: 100−88=12% Expectedpopulation size after TBHP+PC: 100−58−12=30%. Recovered survival: 60%Survival gain in %: 60−30=30 Protection exerted: 30:60×100=50%

4. Estimation of Cytotoxicity by LDH Assay:

For this experiment IMR-32 cells were grown in MEM medium withoutI-Glutamine, Phenolphtaleine and sodium pyruvate 3000 IMR-32 cell/wellseeded in 96 well plates. 24 hours after the oxidative stress, the cellswere washed 2 times with PBS and two rows each were treated with 1:1000and 1:10000 PC-FRTS/1 for 1 h at 37° C. After 1 hour the cells werewashed with PBS two times and PBS was replaced with the growth medium.Two rows each were treated with 1:1000 and 1:10000 PC-FRTS/1 while tworows each we treated with 25 and 50 uM TBHP respectively. Two rows wereleft untreated as Control. The LDH activity was measured by LactateDehydrogenase Assay Kit provided by Sigma Diagnostics. The colorimetricassay measures the residual pyruvate (substrate of the enzymaticreaction). The basal activity present in the medium alone (in theabsence of Cells) was considered 0% and was systematically subtractedfrom each experimental value. Control untreated cells in two rows werelysed with Triton X-1 00 (0.02%) and were treated as samples with 100%LDH release.

Initial two experiments were done to standardize the procedure, numberof cells used wave length for optimum absorbance, optimum pyruvatesubstrate to be used. It was established that 3000 cells/well, 0.4 ml ofpyruvate, spectrophotometer readings at 440 nm were standards.

FRTS/1 was used as pre-treatment, co-treatment and post-treatment withthe oxidative insult.

Pre-treatment.

The cells were pre-treated with FRTS/1 for 2 h before exposure to TBHPdoses.

TBHP 25 uM 50 uM Cell damages TBHP/CT 58% 70% FRTS/1 1:1000 decrease 12%12% Total expected decrease 70% 82% Expected survival 30% 18% SurvivalTBHP + PC/CT 60% 53% Protection exerted by PC 50% 77%

The cells were treated simultaneously with FRTS/1 and TBHP for 1 hour.

TBHP 25 uM 50 uM Cell damages TBHP/CT 39% 67% FRTS/1 1:1000 decrease  7% 7% Total expected decrease 46% 74% Expected survival 54% 26% SurvivalTBHP + PC/CT 83% 62% Protection exerted by PC 35% 62%

The cells were treated with 3 doses of FRTS/1, 1 hour after to have beenexposed to TBHP doses.

TBHP 25 uM 50 uM Cell damages TBHP/CT 38% 66% FRTS/1 decrease  7%  7%Total expected decrease 46% 74% Expected survival 54% 26% SurvivalTBHP + PC-CT 71% 72% Protection exerted by PC 23% 62%

The <<Cell damages TBHP/CT>> represents the damages caused by theoxidant calculated as a percentage from the survival of untreatedcontrols. The <<FRTS/1 decrease>> refers to the difference in populationsize in controls and FRTS/1 exposed cells. The <<Total expecteddecreases>> sums the difference in population size due to TBHP andFRTS/1 exposure individually. The <<Expected survival>> is 100-totalexpected decrease. The <<survival TBHP+PC/CT>> is the actual survival inthe presence of FRTS/1 diluted 1:1000. The <<protection exerted by PC>>is calculated as reported above.

In the pre-treatment experiment, the effect of FRTS/1 is enhanced whencompared to protection exerted by a post treatment (compare 50% to 23%and 77% to 62%). It strongly suggests that the protective effect ofPC-FRTS/1 is exerted through its antioxidant properties. At low dose (25uM), the protection against the damages caused by the oxidant doubles.

Clearly, the efficacy of PC-FRTS/1 is confirmed as a preventivetreatment due to its antioxidant properties.

The antioxidant properties of FRTS/1 are confirmed in the co-treatmentexperiment. The efficiency of the product is similar to the one reportedfor post-treatment at the highest dose of oxidant and intermediate tothe one obtained following pre- and post-treatment, respectively.

FRTS/1 exerts its protection against apoptosis caused by TBHP during thecourse of TBHP damages.

The strong antioxidant properties of FRTS/1 are confirmed via itsprotective effect on ROS generated in the IMR-32 cells by the oxidantTBHP. Under our standardized conditions, the protective effect averages62%.

The antioxidant effect occurs on pre, co- and post-treatment. (FIGS. 11a and b)

The more the oxidative damages caused by TBHP on the IMR32 cells (asTBHP dosage increases), the more the protection exerted by FRTS/1. Thisis independent from the concentration of FRTS/1 since it shows at 1/1000as well as at 1/10,000 dilutions (illustrated in FIGS. 12A and 12Brespectively and). These results show the dynamism of the presentextract.

The fact that the protection increases with increasing damages indicatesthat the mechanism of action of FRTS/1 may differ from that ofconventional antioxidants as indicated in the studies relative to thechemistry of the reaction:

-   1) the protective effect exerted by FRTS/1 lasts longer-   2) The vitamin E and its analogs are used up at given    concentrations, while they exert their antioxidant effect. It does    not seem to be the case with FRTS/1 as illustrated by the above    chemical reactions shown in above.

The correlation between extent of oxidative damages and protection byPC-FRST/1 is a unique property shown by this antioxidant.

Increasing the cell density from 1000 to 3000, obviously decreases thedose of product that each cell receives. This is a well-known effect intoxicology and pharmacology. The protection exerted by FRTS/1 remainsexcellent even when the number of cells to be protected triple. Theeffect is reproducible.

Estimation of Apoptosis in IMR32 Cells by Lactic Deshydrogenase (LDH)Assay:

The classical LDH assay measures the release of the LDH enzyme byapoptotic cells. The assay used in the present study measures theresidual level of enzyme substrate (pyruvate) using a colorimetricreaction. The more the substrate in the medium, the less enzymereleased. Medium cells was taken as 0% enzyme activity released, andlysed cells in the medium as 100% released. The table values arecalculated from these two parameters.

Level of Apoptosis Measured by Release of LDH by the Damaged Cells:

PC-FRTS/1 doses 1:1000 1:10 000 TBHP concentrations 25 uM 50 uM 25 uM 50uM LDH release/TBHP 64% 92% 64% 92% LDH release/PC 32% 32%  0%  0%Expected LDH releases 96% 124%  64% 92% Observed LDH TBHP + 49% 73% 30%66% FRTS/1 Release

The total release of LDH by TBHP plus FRTS/1 individual exposures wascompared to the observed LDH activity (last row).

The protective effect of FRTS/1 is obvious. A post treatment by FRTS/1protects effectively the IMR-32 cells against apoptosis induced by theROS generated by TBHP.

Conclusion

FRTS/1 compound exhibits potent antioxidant properties as assessed bychemical assays.

These highly efficient antioxidant protective effects are also exhibitedin a biological in vitro assay:

-   -   FRTS/1 antioxidant properties demonstrated chemically are        confirmed biologically;    -   FRTS/1 exhibits highly protective effects against ROS damages        causing apoptosis in IMR-32 cells following TBHP exposure;    -   FRTS/1 presents the unique property to be dynamic so to exert        higher protective effect as oxidative damages increase (the        higher the damages by ROS, the higher the protection by FRTS/1);    -   FRTS/1 exhibits a long lasting (hours) anti-oxidative effect        which shows a great level of stability and/or a capacity to        regenerate, which is unique to this antioxidant;    -   FRTS/1 is efficient at doses that are not toxic.        Interaction Between Cells:

To be an effective therapeutic medication, the present extract mustfulfil at least some characteristics. Amongst these, the extract not betoxic, immunogenic or hinder the normal tissue function, particularlythe oxygen and carbon dioxide erythrocyte transport.

The extract should not stick to erythrocytes, although they shoulddisperse in the recipient body to target a tissue or organ to betreated. We have verified if macrophages, which are first line defencecells do not destroy the present extract. Macrophagic mode ofdestruction is normally production of free radicals which destroy bigparticles before phagocytosis. Upon phagocytosis, macrophages producecytokines which are molecules signaling the presence of any intruder orthe malfunctioning of a tissue or cell. Cytokines are molecules sent toother cells, signalling the presence of intruders or of a malfunctioningof a tissue. The cells responding to cytokines are cells likefibroblasts, endothelial cells, macrophages, lymphocytes, neutrophilsand eosinophils. These cells are involved in the process of destructionand/or reconstruction of tissues and organs. These processes involveinflammation. If the response is disorganized or if the intruder iscontinuously present in the organism, the latter suffers of a chronicaldiseases such as rheumatism, cutaneous irritation, conjunctivitis,alveolitis, asthma, and even cancer. The interaction between cells andFRTS/1 were performed with the FAGS technique. This apparatus detectfluorescence of cells (autofluorescence) and also all fluorescentmolecules fixed around. FRTS/1 is a autofluorescent complex, so we canquantify interactions without any modification.

Conclusion: FRTS/1 adhere to macrophages (commercial lines: NR-8383 byATCC) at 37° C. with comparison with mastocytes (positive control, RCMCprovided by ATCC). Macrophages phagocyte FRTS/1 (⅓) after 2 hours, whichdemonstrates that FRTS/1 stays for a rather long period in blood flow.Slow phagocytosis is not a bad news, per se, since another type ofbeneficial effect could be observed consequent to the cytokin activation(“phase II” effect). Since macrophages phagocyte the extract and sinceIMR-32 cells appear to allow entrance to the extract into the cytoplasm,it is believed that the extract may enter the cells by endocytosis.

Oxidative Ex Vivo Models:

Liver perfusion model and brain perfusion model are good experimentalmodels responsive to an oxidative stress. They were used to demonstratethe protective effect of the extract towards vital organs.

Liver Perfusion Model:

A plurality of hepatic functions have been evaluated. The method used toperfuse the liver was described by Drouin et al. 2000 and by Lavoie etal. 2000. Glucose, lactate, ALT and LDH were determined byspectrophotometry, while bile production was simply measured byvolumetry. When compared to a control vehicle, there was no deleteriouseffect observed with the perfusion of the extract. On the contrary, whenthe potency of the extract to reduce the expression of a stress createdby ishemia followed by re-perfusion (I/R), the results were that theextract had a protective effect against oxidative damages induced in theliver.

In this model, the liver is perfused in situ with a controlledextracorporeal circulation, which isolates the liver while preservingits vascular bed intact and saving its structural and functionalintegrity. This model is well documented (Ross 1972) and allows thestudy of oxidative stress as well as of cell damage (Bailey 2000).

Effect on liver viability is undertaken to evaluate the effect FRTS/1 onliver viability. More specifically, the effect of FRTS/1 on hepaticfunctions during in situ perfusion is evaluated.

The livers of Sprague-Dawley rats are perfused in situ with asingle-pass system in a standard Krebs-Henseleit (K-H) solution. [pH7.4, O₂:CO₂ (95%:5%)] The K-H solution is composed of: NaCl (118 mM),KCl (4.8 mM), KH₂PO₄ (1.2 mM), MgSO₄.7H₂O, CaCl₂ (1.5 mM), NaHCO₃ (25mM) and albumin (2% w/v).

Under anesthesia (pentobarbital; 50 mg.kg⁻¹ body weight), a laparatomyis performed to expose the portal vein, the inferior vena cava and thebiliary channel for canulation. The portal canulation is used as theentrance of the perfusate into the liver and that of the vena cava isused for recovering the perfusate at the liver exit. The nervus vagus issectioned to isolate the liver from any vasomotor influence. The totalsurgical procedure is completed within 15 minutes. The time intervalbetween the insertion of a canula into the portal vein and the beginningof the circulation is not more than 3 minutes. The time lapsed betweenthe cardiac arrest caused by the thoracic cage opening and the beginningof the perfusion is not beyond one minute.

The total duration of the perfusion was 60 minutes. During the first 30minutes, a wash out was performed. During that period, the K-H solution(37° C.), was supplemented with glucose (8 mM), lactate (0.5 mM),alanine (0.2 mM) and glycerol (0.02 mM) was circulated in the liver inan open circuit. Thereafter, the liver was exposed to vehicles only(control), or to FRTS/vehicles (treated FRTS/1 group), for another 30minutes. The vehicles comprise either saline (1 ml/800 ml perfusate), or1,3-propanediol (24 ml/800 ml perfusate). FRTS/1 was added to eitherpropanediol (0.06 mg FRTS/ml propanediol:24 ml/800 ml perfusate) or tosaline (2 mg FRTS/ml saline: 1 ml/800 ml perfusate). The perfusate flowrate was kept constant at 6 ml/minute/100 g body weight). A small sampleof the perfusate was taken at the entrance and at the exit of the liver,for determining the production of the utilization of metabolites.

The concentrations of glucose, lactate, ALT and LDH in the perfusatewere determined by photospectrometry using commercial proceduredisclosed by Sigma-Aldrich Canada n. 17-UV, No. 735, No. 59-UV and No.DH1240-UV, respectively). The extraction or the production of asubstrate by the liver is measured by the differences between theentrance and the exit of a metabolite, multiplied by the perfusate flowrate.

Since the nature of the vehicle did not influence the measuredparameters, the results have been combined.

The bile production was similar in both control and treated groups(0.55±0.10 v. 0.62±0.12 mg/min/g liver, in the control and treatedgroup, respectively). During reperfusion, bile production diminushedwhen compared to the pre-ischemia levels in both treated and controlgroups. Bile production returned to normal levels within 10 minutesafter reperfusion.

At the entrance, the glucose concentration was similar in both groups(7.04±0.40 v. 7.24±0.07 mM in control and treated group, respectively).In both groups, the liver has slight tendency to use glucose. Theexposure to FRTS/1 has no effect on glucose capture (0.32±0.21v.0.39±0.25 μM/min/g liver in control and treated group, respectively).The concentration of lactate at the entrance was also similar in bothgroups (0.60±0.33 v. 0.50±0.10 mM in control and treated group,respectively). Upon exposure to FRTS, there is a slight tendency in thetreated livers to produce lactate (−0.01±0.1 v. 0.40±0.005 μM/min/gliver in the control and treated group, respectively).

Perfusing the liver by itself does not provoke any release of ALT or LDHin the control groups (−0.07±0.14 and 1.79±0.47 μM/min/g, respectively).Treatment with FRTS/1 does not appear to provoke a release of ALT or LDHby the liver (0.57±0.21 and 2.36±1.10 μM/min/g), although there is aslight tendency to increase. When liver were partly perfused before theuse of FRTS, there was a progressive increase of the LDH production bythe liver (35.57±8.96 μM/min/g).

These preliminary results lead to believe that FRTS/1 has no remarkableeffect on the viability of perfused liver. More specifically, treatmentwith FRTS/1 do not appear to modify the liver functions during a 30minute perfusion duration, as evaluated by the utilization of glucose,the production of lactate and of bile. There is no apparent structuraldamage to the hepatocytes, since there is no increase of ALT and LDH.

The above was slightly modified to study the effect of ischemia andreperfusion. The total duration of the perfusion was 105 minutes. The31^(st) minute constituted a wash out period. During that period, a K-Hsolution (pH 7.4 and in the presence of O₂:CO₂ 95%:5%) was added toglucose 8 mM, lactate 0.5 mM, alanine 0.2 mM and glycerol 0.2 mM. Thesolution was circulated in an open circuit. Then, the liver was exposedto the extract or to the vehicle: 1,3-propanediol for 15 minutes. Theperfusing rate was kept constant at 6 ml per minute per 100 g of bodyweight (Drouin et al 2000, Lavoie et al. 2000). The perfusion wasstopped for 30 minutes. During this arrest, ischemia developed. Thencirculation was re-established for a duration of 30 minutes. Theperfusing liquids were taken at the entrance and at the exit of thetested livers, for measuring the production or the use of the evaluatedbiological markers.

Upon re-perfusion, the bioproduction diminishes compared to thepre-ischemia levels in both control and treated livers. These levelsreturn to normal within 10 minutes after the beginning of there-perfusion. Control and treated livers release glucose in identicalway (10.4±0.7 μM.minute⁻¹.g⁻¹). Glucose production was however slightlysuperior after 80 minutes in the treated group, compared to the controlorgans. Lactate accumulated during ischemia in higher levels in treatedlivers (5.9±0.5 μM.minute⁻¹.g⁻¹). when compared to the control organs(4.5±0.1 μM.minute⁻¹.g⁻¹).

Pre-treatment with the extract decreases the release of ALT (1.09±0.44mU.minute⁻¹.g⁻¹vs. 2.44±0.79 mU.minute⁻¹.g⁻¹, respectively) duringre-perfusion. Pre-treatment with the extract appears to diminish theimpact of ischemia for the first 30 minutes of re-perfusion. Ischemiawithout any pre-treatment with the extract, provokes an increase of LDH(108.7±27.3 mU.minute⁻¹.g⁻¹) during re-perfusion. Pre-treatment with theextract does not influence LDH in increase (115.9±60.8 mU.minute⁻¹.g⁻¹)

Potassium and sodium plasmatic concentrations were also measured in bothgroups. At the beginning of the re-perfusion, the release of potassiumin treated group is superior to the control (0.43±0.01 mU.minute⁻¹.g⁻¹vs5.4±0.1 mU.minute⁻¹.g⁻¹, in control and treated groups, respectively).In the perfusate, at the entrance, the plasmatic concentration ofpotassium and sodium were similar in both groups. (K⁺=5.6±0.3 mM and5.4±0.1 mM in control and treated groups, respectively; Na⁺=142.2±8.6 mMand 137.2±15.2 mM, in control and treated groups, respectively).

At the beginning of the re-perfusion, the release of sodium was alsosuperior in treated groups when compared to the controls (1.2±1.1μM.minute⁻¹.g⁻¹ and 16.5±3.9 μM.minute⁻¹g⁻¹, respectively).

Ischemia followed by re-perfusion is characterized with circulatory andmetabolic disturbance, and with tissue damage provoked by free-radicals(Lee 2000). This particular model is currently used to evaluate thedamage provoked by free radicals in tissues or organs subject totransplantation (Smrekova 2000, Cohen 2000 and Hachimoto 2000).Structural and functional disturbance is occasioned by I/R and arereflected by an increase in the release of enzymes (Bailey 2000, Vollmar1994 and Perlata 1999), a decrease of bile production (Vollmar 1994) anda depletion of ATP reserve (Hwang 1999 and Amllet 1990). Ischemia maylead to the production of singlet oxygen, peroxide, and superoxide (O₂⁻., H₂O₂, OH.) following the release of metal ions like iron and cupper(Halliwell 1999). This model is therefore a good one for characterizinga protective effect, if any, which would be present in the presentextract following a radical insult.

Metabolic changes insuring protection of hepatic cells are affected bypre-treatment with the extract. A radical attack consequent to I/Rprovoke decrease in ATP cell content, thereby modifying the energeticstate of the cell (Peralta 2000, Mallet 1990). Tamarina et al. (1984)suggest that ischemia damages the glycolytic system of a cell, whichrenders difficult the lactate production. A healthy cell (or a cellunder the action of a protecting agent) would protect itself from such astress by increasing its ATP production via the glycolitic pathway. Sanoet al. (1995) proposed that glycolytic activation reduces I/R-inducedfree-radical formation. Also phosphoenolpyruvate prevents the ATPdecrease consequent to I/R (Saiki 1999). Hwang et al. (1999) suggestthat a decrease of the ratio NAD⁺/NADH is essential to the resistanceagainst free radicals to minimize cell damages. Pre-treating the cellswith the present extract increases lactate production during theischemic period, which apparently reflects the activation of a defensemechanism against the decrease in ATP provoked by ischemia. Kowalski1992 and Groussard 2000 suggest that lactate could play a protectiverole in ischemia. Lactate could buffer superoxide (OH.), generatingpyruvate which also buffers peroxide and superoxide, while decomposinginto acetate and CO₂ (Herz 1997).

The potassium exit of a cell pre-treated with the present extract alsosupports a protective effect of the extract on a cell. Membranecomponent peroxidation may damage potassic channels (Halliwell 1999).The potassium release may be a beneficial adaptation against a metabolicstress (Wang et al. 1996). Potassium channel opening would permitcapturing substrates for the intracellular ATP generation (Wondergem1980). Potassium releasing would also inhibit HCO₃ ⁻ transport,contributing to the acidification of the cytoplasm, which is alsoprotective to the cell (Currin 1991). Potassium release appears early ina perfusate and precedes the ALT release, and is proportional to the ATPdecrease (Mets 1993).

Sodium accumulation in the cell plays a major role in cell damageinduced by I/R (Carini 2000, vanEchteld 1991 and Xia 1996). Thisincrease may be due to: 1) a dysfunctional Na⁺/K⁺ pump, which is due toa decrease in ATP and to an increase of inorganic phosphate, or 2) astimulation of the Na/H anti-carrier, due to the acidification of thecytoplasm (Zhao-fan 1996). Any reversal of such a situation coulddecrease the impact of a metabolic stress induced by sodium accumulation(Fiegen 1997). The sodium release may therefore be a defense mechanismagainst damages. Both decrease of potassium and sodium have beenobserved with livers perfused with present extract, which supports theprotective effect of the extract in hepatic cells. Sodium release issuperior in the treated group when compared to the control group, 10minutes after re-perfusion, which suggests a late recovery in aTPcontents in the controls.

The present extract stimulates the cellular mechanisms associated withcell protection against a radical attack.

The ex-vivo Effect of the Present Extract in Brain:

Numerous brain pathologies involve an increase production of freeradicals. The latter are believed to contribute to the neurodegeneratingprocess, namely pursuant to cerebral vascular accidents, or during thedevelopment of Alzheimer disease. It is believed that anti-radicalcomponents may be useful for reducing the expression ofneurodegenerative diseases, or for their prevention or their treatment.

Hyppocampus region of the brain is vulnerable to neurotoxic effect offree radicals, namely during a cerebral anoxia. It has been demonstratedin vitro that neuronal transmission is attenuated during anoxia, becauseof the over expression of antioxidant molecules. Therefore, the effectof the present extract on the hyppocampal neuronal transmission wasevaluated. Electrophysiological responses were registered in this brainstructure. Particularly, the recovery of neuronal potentialisation wouldbe studied. Hyppocampal slices of 450 μm thickness were obtained fromrat brains and were transferred to electrophysiological chambers. Theslices were maintained during 60 to 90 minutes in an oxygenatedphysiological solution, comprising or not different concentration or notdifferent concentrations of the extract. After this resting period,electrical stimulation were applied every 25 seconds in hyppocampalafferences (Schaffer, regions of CA1 region). These electricalstimulations evolved synaptic responses. The initial slope ofpost-excitatory potentials was calculated to quantify the synaptictransmission efficacy pursuant anoxia. The study the neuronalpotentialization, a high frequency stimulation train was applied to theneuronal circuits after anoxia. As a positive control, the response toglutamate with or without ischemia may be measured and the presence ofthe present extract should restore the response to glutamate.

Toxicity of FRTS/1

The toxicity has been evaluated in two different models: the in situliver perfusion and in situ brain perfusion. These organs did not showany sign of toxicity due to the presence of the extract.

Adverse Reactions

The Present Extract is Non-immunogenic.

The immune response provoked by the present extract has been evaluatedby injecting 125 μg of the extract intraperiteonally in mice three timesat 7 day intervals. After a week spent following the last injection,blood was harvested for immune serum obtention. The mice wereanesthetized and the blood was taken by cardiac puncture. Blood sampleswere put immediately on ice. Blood clotting was allowed to proceed onice and samples were centrifuged 12 hours after harvesting. Thecentrifuging conditions were the following: 10 minutes at 2500 g, whichprovided about 500 μl of serum. The extract was adsorbed on a microplateto provide a fixed antigen preparation. 200 μl of the extract (5 μg/ml)and 200 μl of ELISA buffer were poured in the wells of a 96-well plate.The ELISA buffer was made of 100 mM of sodium carbonate buffer (pH 9.6).After incubation of the antigen at 4° C. overnight, the non-adsorbedantigen was eliminated three washing steps with a sodium phosphatebuffer 100 mM/NaCl, 100 mM (pH 7.4). The free adsorption sites wereblocked in a 60 minute-incubation at room temperature with a solution ofcaseine 3% in the sodium phosphate/NaCl buffer. Excess of caseine waswashed three times. The serum samples providing the antibodies, if any,were prepared as follows: serial dilutions in the sodium phosphate/NaClbuffer supplemented with 0.05% Tween 20™ were made and 25 μl of thesedilutions were added to the wells. The microplate was further incubatedfor 1 h at 37° C., which step was followed by 5 washes with the sodiumphosphate/NaCl/Tween buffer.

The presence of an antibody was revealed by the formation of ananti-Ig-peroxidase complex. 25 μl of the enzyme dilution in the sodiumphosphate/NaCl/Tween buffer is added to each well followed by a onehour-incubation at 37° C. The enzyme dilution varied between 1:750 and1–3000, depending on the conjugate (These are anti-Ig goat anti-sera,IgM, IgG, Ig1, Ig2 and Ig3, labelled with peroxidase). The labellingstep was followed with 5 washings with the buffer. Substrate hydrogenperoxide 0.015% and the chromogene ABTS (2,2azino-diethylbenzthiazoline-6-sulfonate) 0.05% dissolved in a phosphatecitrate buffer 100 mM (pH 4.0) were added to the wells. Another 30minute-incubation at room temperature and in the dark was allowed toproceed. The action of the enzyme released a colored substance which canbe read spectrophotometrically at an absorbance wavelength of 405 nm.The serum level of antibody specific to the extract should beproportional to the color intensity. The results that have been obtainedindicate that the extract is non-immunogenic to the recipientindividuals.

These results indicate that the extract is not toxic to individuals,since it is neither hepatotoxic nor immunogenic.

Compositions and Dosage Regimens:

Due to the stability and the potency of the present extract and the factthat it is non-toxic to animals, it is believed that dosage ratesextending from 1 ng per kg of body weight to 1 g per kg of body weightper day could be administered to individuals in need for suchadministration (a dose in a lower μg range may be preferred). The dosagedepends on the agressivity sought for the treatment of a disease. Thedosages may also depend on the formulations and their route ofadministration. For example, a topical composition will not comprise thesame dose as an intravenous composition or an enteral composition.

Topical Compositions for Treating Skin or Mucosal Diseases:

Allergy/Asthma:

Brown Norway rats are high IgE producers. There is a well establishedmodel of allergen-induced airway hyperresponsiveness in Brown Norwayrats¹ that reflects many features of human allergic asthma, includingboth early and late (70% of animals) phase reactions, increase inantigen-specific IgE following active immunization,² airwayinflammation,³ and increased bronchial responsiveness to several stimulifollowing allergen challenge.⁴

Measurement of pulmonary responses. Brown Norway rats are sensitized byintraperitoneal injection of 1 ml of 1 mg ovalbumin/100 mg Al(OH)₃ insaline as previously described.⁵ Twenty one days later, animals areanesthetized and intubated as previously described⁶ with the end of theendotracheal tube connected to the Plexiglas box. A water-filledoesophageal catheter attached to a pressure transducer is used todetermine changes in pleural pressure. Airflow is measured by apneumotachograph coupled to a differential transducer attached to thePlexiglas box. Air flow, volume and transpulmonary pressure, pulmonaryresistance (R_(L)) are determined at different times to identify boththe early and late phase reactions. Sensitized Brown Norway rats arechallenged with saline or ovalbumin (2% in saline) using the “Wright”nebuliser from Roxon Medi-Tech Lté (Montréal, PQ) using compressed airwith a pressure giving an output of 0.1–0.2 ml/min passed into thePlexiglas box. Pulmonary resistance is measured every 5 minutes for thefirst hour and every 15 minutes for the next 10 hours. Pre-, co- andpost-treatment with the extracts administered i.p. or by inhalation(about 1 to 100 μg) are tested in this model to provide improvement.

Protection Against UV Radiation

The ability of FRST/1 to prevent or reduce the UV-induced skin damagesin hairless mice was investigated. As it is known that most of the skincancers are induced by exposure to UV radiation, there is a need toidentify new potent natural compounds that could prevent the adverseeffects of UV radiation.

Animals

Hairless albino (SKH/1) mice will be purchased from Charles Riverlaboratories (Wilmington, Mass.).

All mice are 6 weeks old at the beginning of the irradiation period.Mice are housed and maintained under standard conditions (23±10 C.,42±6% relative humidity, 12:12-h light-dark cycle) at the AnimalFacility of IBS. Lights are automatically switched on daily at 7 AM andswitched off daily at 7 PM. Mice are fed Purina chow diet (24% protein,4% fat, and 4.5% fiber) and water ad libitum. For irradiation, mice areplaced in plastic cages and are allowed to move freely within the cagesduring irradiation.

The animals were acclimated for one week prior to treatment and dividedinto randomly into 5 groups as follows.

-   Group I: Control non-irradiated, non-treated (n=5);-   Group II: non-UV-irradiated animals treated with a preparation of    topical ointment containing FRST/1 (n=5).-   Groups III: UV irradiated animals treated with the preparation of    topical ointment without FRST/1 (n=5)-   Group IV: animals receiving topical application of the cream    containing FRST/1 during UV irradiation (n=5).-   Group V: non-treated UV-irradiated animals (n=5). Treatments consist    in:-   1. Weighing all animals to assess their health on the day of the    treatment and once every second day thereafter.-   2. Performing a dorsal topical application of one type of cream    (known to be non-toxic to animals and human). The cream contains or    not FRST/1. The extract is present at a 1:10,000 dilution (starting    from the lyophilized thylakoid fraction). One gram of cream is    dispensed just before (and remain during and after UV irradiation)    on the back of 3 groups of animals. Among these, one group of 5    animals receives the cream and no irradiation. One group receives    the cream without FRST/1 and is exposed to UVB and one group    receives the FRST/1 cream and UVB. The UVB irradiated groups (n=5)    are exposed to sunlamps for 10 min once. One group (n=5) is exposed    to UV sunlamps without cream treatment.-   3. A single 10-min UVB irradiation by sunlamps located at 60 cm from    the animal backs is performed. The two groups of 5 animals tested    for cream protection (with and without FRST) and a single group of 5    animals without protection are submitted to the single dose of UVB.-   4. After treatment, all animals are kept in cages and photographed    from the top of the cage on the day of UV treatment and 3 to 4 days    later, without manipulation.-   5. After one week, the animals are sacrificed before removing a    piece of their dorsal skin for further studies.

Westinghouse FS40 sunlamps, an IL-1400 radiometer, and a UVB photometerare used. The spectral irradiance for the UV lamps is 280–400 nm, 80% ofwhich are in UVB region and 20% in UVA region. The peak intensity of thelight source are 297 nm. The fluence at 60 cm from the dorsal surface ofthe mice are 0.48–0.50 mJ/cm2/s. The mice are placed in plastic cageswithout lid as mentioned above.

Negative control mice (Groups I, II,) are treated in an identical way,but UV lamp is not be switched on. Group II receives the ointmentwithout FRST/1 topically.

The mice from Group III, IV and V are given a single exposure of a totalof 200 mJ UV light/cm 2 (acute dosage) for 10 min. In Group IV theanimals receive topical application of a FRST/1 preparation immediatelybefore, during and after UV exposure. This approach has been adopted totake into account minor differences in UV absorption characteristics(optical density differences) in the 290–320 nm range that mightoptically influence the UV light irradiation condition (Gonales andPathak 1996).

The mice are kept and weighed every second day one week before and oneweek after UV irradiation. At the end of the experiment, the mice aresacrificed and the following parameters are compared in all the groups.

At the end of the experiment, the mice will be sacrificed and thefollowing parameters will be compared in all the groups.

-   1. Body weight-   2. Epidermal observation and photographs-   3. Following sacrifice of the mice, a piece of the dorsal skin is    surgically removed and used for further analyses.-   4. Comparison of Cytokeratin patterns of expression by quantitative    Western blot.

Solar radiation is the major environmental factor that affects thestructure and function of human skin. Long term cutaneous photodamage asa consequence of cumulative UV radiation injury often leads tophotoaging and skin cancer in fair-skinned individuals. Studiesinvolving photobiological effects of ultraviolet radiation reveal thatthe ultraviolet B (UVB) component (290–320 nm) in particular iserythemogenic, carcinogenic and induces skin photoaging changes precededby direct damage to DNA, RNA, proteins (including enzymes), cellmembrane and other cell organelles; Tedesco,et. al. (1997).

Kligman and Kligman (1993) made a clear distinction betweenchronological aging and photoaging. Photoaging is used to describe theclinical and histological damages produced by chronic exposure of theskin to sunlight or solar-simulated UV radiation. Histologically, thesechanges are manifested in the form of marked changes in elasticity,glycosaminoglycans, and disordered collagen, together with an increasein the number of mast cells and inflammatory cells (Kligman andGebre,1991; Kaaresn and Poulsen, 1995; Poulsen et. al., 1984). Thisphenomenon of photoaging has clinically been recognized as irreversible,although recent therapeutic approaches have helped to minimize the overexpression of intrinsic aging and photoaging changes (Gilchrest, 1996;Kang and Voorhees, 1998). The use of sunscreens on a regular basis hasbeen reported to help prevent actinic damage to connective tissue(Snyder and May, 1975; Kligman and Kligman, 1982; Bissett et. al. 1991).The use of both sunscreens and antioxidants (e.g., green tea, Vitamin C,Vitamin E) appears to have an inhibitory effect on UVB-induced acuteskin damage that contributes to both photoaging and photocarcinogenesis(Synder and May, 1975; Bissett et. al., 1991 and Huang et. al. 1997).

The photoprotective ability or antioxidant effect of FRST/1 againstacute UV radiation exposure has been evaluated in the hairless albinomouse model.

The skin of hairless albino mouse (SKH-1) has been recognized as auseful and relevant experimental model for studying and understandingeffects of UV radiation and photoaging of human skin (Kligman et.al.,1989; Bissett, et. al., 1987; Chatterjee et. al., 1990; Kligman andGebre, 1991). The visually and microscopically recognizable responses ofthe epidermis and of the dermis to UVB radiation and absence of hairmake the skin of hairless mice particularly useful in studying andevaluating the damaging effects of UV radiation. This mouse model hasalso been used to study and examine the immunological alterations andcarcinogenesis induced by UVB radiation both locally in the skin andsystematically (Fisher, et. al.,1989; Ho, et. al., 1992; Reeve, et. al.,1991).

Results:

Right after irradiation, all the non-treated irradiated mice showedsigns of skin irritation and itchiness. They were otherwise healthy andactive, although they did not gain weight. No symptoms of irritation orredness was observed on irradiated mice when pre-treated as well as whenpost-treated with FRST/1 and mice gained in weight in 80% of cases.Therefore, topical compositions, either solar screen lotion, cream,ointment, oil, gel or spray, are objects of this invention.

Skin Cytokeratins Analysis:

The cytokeratins 1 and 10 are representative of mature skin. A decreasein these keratins is an indication of epidermis regeneration followinglesion.

The cytokeratins 5 and 8 are representatives of suprabasal and basallayers and are supposed to be expressed in actively proliferatingepidermis.

The presence of these cytokeratins was evaluated with specificantibodies.

The extracts from mice skin were individually extracted and the skin ofmice was analyzed individually per group. The same amount of totalextracted cytoplasmic proteins was applied per well: 35 ug to enablecomparison among animals and treatments.

Conclusions:

FRST/1 protected mice showed a pattern similar to that of controlsuntreated non-irradiated, while all other treatments showed a drasticdecrease in high molecular weight keratins. Especially, cytokeratin 10remained very well expressed in the skin of FRST/1 mice, which is a goodindication of a protective effect. At a very low dose, the extract wasactive topically. The dose may be increased at will since the dose ofthe extract is not limited by any toxicity.

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified, without departingfrom the spirit and nature of the subject invention as defined in theappended claims.

TABLE 1 Relative activity in function of the species and the exogenfluid. Exogen fluid/ Relative Activity 67 g of plant Species 0.1 mlSpinacia oleracea 1.12 193 Common name: Spinach 0.84 0 1.11 100 1.1 300Lycopersicon esculenta 0.54 193 Common name: Tomato Capsicum annuum 0.4193 Common name: Green pepper Lactuca sativa Romaine 0.83 193 Commonname: Romaine lettuce Brassica oleracea capitata 0.95 193 Common name:Cabbage Hordeum spp. 0.29 193 Common name: Barley Lactuca sativa (greenice) 0.93 193 Commmon name: Green ice lettuce Lactuca sativa (Boston)0.97 193 Common name: Boston lettuce 0.8 0 1.5 100 0.95 300 CrassulaArborescens 0.71 193 0.02 0 0.37 100 0.67 300 Picea mariana 0.7 400Common name: Black Spruce

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1. A method of obtaining an extract from a photosynthetic organism, saidmethod comprising the steps of: a) providing a suspension of organismconstituents that contain thylakoids; b) disrupting the constituentswhile recovering thylakoids under light conditions which minimize lightflux, in a medium having a viscosity comprised between 1 to 1.3centipoise and a pH above 2 and below 10; the medium being added in avolume calculated upon the following equation:$\frac{\left( {{{Volume}\mspace{14mu}{of}\mspace{14mu}{medium}} + {{organism}\mspace{14mu}{constituents}\mspace{14mu}{water}\mspace{14mu}{content}}} \right)}{\left( {{Organism}\mspace{14mu}{constituents}\mspace{14mu}{dry}\mspace{14mu}{weight}} \right)} > 10$whereby a first extract essentially constituted of thylakoids, cellsdebris/membranes, and a liquid phase is obtained, said thylakoidscomprising organized photosynthetic pigments; c) separating thylakoids,cell debris/membranes and liquid phase from each another, to form asecond, third and fourth extracts essentially constituted by isolatedthylakoids, cell debris/membranes, and a liquid phase, respectively; andd) eliminating any electron donor from said first, second and thirdextracts.
 2. The method according to claim 1, wherein the equation is:$\frac{\left( {{{Volume}\mspace{14mu}{of}\mspace{14mu}{medium}} + {{organism}\mspace{14mu}{constituents}\mspace{14mu}{water}\mspace{14mu}{content}}} \right)}{\left( {{Organism}\mspace{14mu}{constituents}\mspace{14mu}{dry}\mspace{14mu}{weight}} \right)} = {25 - 150.}$3. The method according to claim 1, wherein the pH is between 5 and 8.4. The method according to claim 1, wherein the pH is between 6 and 7.5.5. The method according to any one of claims 1, 2, and 4, wherein saidorganism is a plant.
 6. The method according to claim 5, wherein thesuspension of step a) is obtained by mechanically dispersing plantconstituents or tissues in said medium.
 7. The method according to claim5, wherein step a) is preceded by a step of submitting a plant to aconditioning parameter selected from light, osmotic stress, heat, cold,freezing, dryness, hormones, chemical and biological inducers.
 8. Themethod according to claim 1, wherein step a) is preceded by a step ofconditioning said organism in a light environment of a wavelengthcomprised between about 500 and 600 nm, and step b) is performed underthe same light conditions.
 9. The method according to claim 5, whereinstep a) is preceded by a step of conditioning said plant in a lightenvironment of a wavelength comprised between about 500 and 600 nm, andstep b) is performed under the same light conditions.
 10. The methodaccording to claim 1, wherein said viscosity is partly achieved byadding a sugar in the medium.
 11. The method according to claim 1,wherein said viscosity is partly achieved by the presence of sorbitol ina concentration of about 0.2 to 1.5 M in said medium or of a sugarachieving a viscosity equivalent to 0.2 to 1.5 M sorbitol.
 12. Themethod according to claim 1, wherein said viscosity is partly achievedby the presence of sorbitol in a concentration of about 0.2 to 0.4 M insaid medium or of a sugar achieving a viscosity equivalent to 0.2 to 0.4M sorbitol.
 13. The method according to any one of claims 1 to 4, and 6to 12, wherein said medium has the following composition: Tris oracetate or ascorbate buffer (20 mM) having a pH of 7.5 and sorbitol orsucrose or fructose 350 mM.
 14. The method according to any one ofclaims 1 to 4, and 6 to 12, wherein the step of separating is performedupon a difference of sedimentation coefficient of each of thylakoids,cell debris and membranes, and liquid phase.
 15. The method according toclaim 14, wherein the step of separating comprises centrifuging the fiatextract in a tube equipped with a filter in a superior portion of thetube, the filter having a porosity onto which cell debris and membranesdeposit while the thylakoids and the liquid phase pass through thefilter, the thylakoids forming a pellet in an inferior portion of thetube.
 16. The method according to any one of claims 1 to 4, 6 to 8, and10 to 12, wherein said electron donor is water.
 17. The method accordingto claim 16, wherein water is eliminated under vacuum freeze drying. 18.The method according to claim 16, wherein water is eliminated byexchanging it against an amphoteric solvent or surfactant after step c).19. The method according to claim 16, wherein water is eliminated byexchanging it against propylene glycol.
 20. The method according toclaim 5, wherein said viscosity is partly achieved by the presence ofsorbitol in a concentration of about 0.2 to 0.4 M in said medium or of asugar achieving a viscosity equivalent to 0.2 to 0.4 M sorbitol.
 21. Themethod according to claim 5, wherein said medium has the followingcomposition: Tris or acetate or ascorbate buffer (20 mM) having a pH of7.5 and sorbitol or sucrose or fructose 350 mM.
 22. The method accordingto claim 5, wherein said electron donor is water.
 23. The methodaccording to claim 9, wherein said electron donor is water.
 24. Themethod according to claim 22, wherein water is eliminated under vacuumfreeze drying.
 25. The method according to claim 23, wherein water iseliminated under vacuum freeze drying.
 26. The method according to claim22, wherein water is eliminated by exchanging it against propyleneglycol.
 27. The method according to claim 23, wherein water iseliminated by exchanging it against propylene glycol.
 28. A method ofobtaining an extract from a plant, said method comprising to steps of:a) exposing the plant to a light environment of a wavelength comprisedbetween about 500 and 600 nm, b) providing a suspension of plantconstituents that contain thylakoids; c) disrupting the constituentswhile recovering thylakoids under the light environment of step a), in amedium having a viscosity comprised between 1 to 1.3 centipoise andcomprising Tris or acetate or ascorbate buffer (20 mM) having a pH of7.5, and sorbitol or sucrose or fructose 350 mM; the medium being addedin a volume calculated upon the following equation:$\frac{\left( {{{Volume}\mspace{14mu}{of}\mspace{14mu}{medium}} + {{organism}\mspace{14mu}{constituents}\mspace{14mu}{water}\mspace{14mu}{content}}} \right)}{\left( {{Organism}\mspace{14mu}{constituents}\mspace{14mu}{dry}\mspace{14mu}{weight}} \right)} = {25 - 150.}$whereby a first extract essentially constituted of thylakoids, cellsdebris/membranes, and a liquid phase is obtained, said thylakoidscomprising organized photosynthetic pigments; d) separating thylakoids,cell debris/membranes and liquid phase from each another, to form asecond, third and fourth extracts essentially constituted by isolatedthylakoids, cell debris/membranes, and a liquid phase, respectively; ande) eliminating any electron donor from said first, second and thirdextracts.